Assay method for group transfer reactions

ABSTRACT

The present invention relates to methods for detecting, quantifying and high throughput screening of donor-products and the catalytic activities generating the donor-products in group-transfer reactions. The invention further provides immunoassays, antibodies and kits that may be used to practice the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/443,746 filed Jan. 30, 2003, which is incorporated byreference here in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

TECHNICAL FIELD

[0003] The present invention relates to group transfer reactionmethodologies. The invention provides methods for the detection andquantification of donor-products and the catalytic activities generatingthe donor-products in group transfer reactions. The invention alsoprovides methods for high throughput screening of acceptor substrates,inhibitors, or activators of enzymes catalyzing group transferreactions. The invention further provides immunoassays, antibodies andrelated kits for practicing the invention.

BACKGROUND OF THE INVENTION

[0004] There are many important biological reactions where thesubstrates are modified by chemical groups that are donated by othersubstrates, known as activated donor molecules. These biologicalreactions are broadly recognized as “group transfer reactions” and havethe general reaction:

donor-X+acceptor→donor-product+acceptor-X.

[0005] Typically, donor-X, the activated donor molecule, is a nucleotideattached to a covalent adduct. The donor-X is activated by formation ofa phosphoester bond in the nucleotide donor. Also the acceptorsubstrates can include small molecules or macromolecules such asproteins or nucleic acids. Products of this reaction are the modifiedacceptor, acceptor-X and the donor-product molecule.

[0006] There are many enzymes that catalyze group transfer reactionssuch as for example kinases, which use ATP to donate a phosphate group;sulfotransferases (SULTs), which use phosphoadenosine-phosphosulfate(PAPS) to donate a sulfonate group; UDP-glucuronosyltransferases (UGTs),which use UDP-glucuronic acid to transfer a glucuronic acid group;methyltransferases, which use s-adenosyltransferase to donate a methylgroup; acetyl transferase, which use acetyl coenzymeA to donate anacetyl group; and ADP-ribosyltransferases, which use nicotinamideadenine dinucleotide (NAD) to donate an ADP-ribose group. Thus, many ofthe enzymes catalyzing group transfer reactions are of interest topharmaceutical companies.

[0007] Automated high throughput screening (HTS) assays are the paradigmfor identifying interactions of potential drug molecules with proteinsin a drug discovery setting, and this format requires simple, robustmolecular assays, preferably with a fluorescent or chemiluminescentreadout. The most suitable format for HTS is a homogenous assay, (e.g.,“single addition” or “mix and read” assay), which does not require anymanipulation after the reaction is initiated and the assay signal can bemonitored continuously. Despite their importance from a drug discoveryperspective, the incorporation of group transfer enzymes intopharmaceutical HTS programs is being slowed or prevented for a number ofreasons: a) homogenous assay methods are not currently available, b) thedetection methods used place serious limits on the utility of the assay,and c) the non-generic nature of the assay requires the development ofmany specific detection reagents to test diverse acceptor substrates.

[0008] The approach currently used to identify sulfotransferasesubstrates or inhibitors requires the use of radioactivity and involvescumbersome post-reaction separation steps, such as precipitation orchromatography. For instance, ³⁵S-PAPS is used in a sulfotransferasereaction and the labeled product is quantified by scintillation countingafter selective precipitation of unreacted ³⁵S-PAPS (Foldes, A. andMeek, J. L., Biochim Biophys Acta, 1973, 327:365-74). This approach isnot desirable in a high throughput screening (HTS) format because of thehigh radiation disposal costs and because the incorporation ofseparation steps complicates the automation process. Other SULT assayshave been developed using calorimetric and fluorescent means, but theyare dependent on the use of a specific acceptor substrate for detection,so their use is limited to a single SULT isoform, and they cannot beused to screen for diverse substrates (Burkart, M. D. and Wong, C. H.,Anal Biochem, 1999, 274:131-7; Frame, L. T., et al., Drug Metab Dispos,2000, 28:1063-8). As a result, SULT interaction studies are currentlynot included during the preclinical development of drugs. Also UGTs arecurrently assayed using radiolabeled donor molecules and requirepost-reaction separation steps such as thin layer chromatography (TLC)or high pressure liquid chromatography (HPLC) which seriously hamperspreclinical HTS programs (Ethell, B. T., et al., Anal Biochem, 1998,255:142-7). Likewise, traditionally, kinases have been assayed by filtercapture or precipitation of radiolabeled polypeptide substrates producedusing 32P-ATP or ³³P-ATP as donor. However, since this method requires aseparation step such as filtering or centrifugation, it cannot be easilyadapted to an automated HTS format. Surface proximity assays (SPA) allowradioassays in a multiwell format with no separation (Mallari, R., etal., J Biomol Screen, 2003, 8:198-204) but their use by pharmaceuticalcompanies is declining because of the disposal and regulatory costs ofhandling radioisotopes.

[0009] Because of the high level of interest in developing kinaseinhibitor drugs, there has been a great deal of effort, scientists todevelop improved assay methods for this enzyme family. Homogenous assaymethods have been developed, in which highly specific reagents are usedto detect the reaction products in the presence of the other componentsof the reaction using a light-based readout, such as fluorescence orchemiluminescence. The most common homogenous approach used for kinaseassays is immunodetection of phosphopeptide products exhibitingdifferent fluorescence properties (Zaman, G. J., et al., Comb Chem HighThroughput Screen, 2003, 6:313-20). In this method, phosphorylation ofsubstrate peptide leads to displacement of a fluorescent phosphopeptidetracer from anti-phosphopeptide and causes a change in its fluorescenceproperties. This basic approach has been adapted to several differentreadout modes used for competitive immunoassays including Fluorescencepolarization (FP) (Parker, G. J., et al., J Biomol Screen, 2000,5:77-88); time resolved fluorescence (Xu, K., et al., J Biochem MolBiol, 2003, 36:421-5); fluorescence lifetime discrimination (Fowler, A.,et al., Anal Biochem, 2002, 308:223-31); and chemiluminescence (Eglen,R. M. and Singh, R., Comb Chem High Throughput Screen, 2003, 6:381-7).

[0010] The shortcoming with this approach is the requirement forphosphopeptide-specific antibodies. Though generic phosphotyrosineantibodies are common, phosphoserine and phosphothreonine antibodies arenotoriously difficult to produce and only recognize phospho-serine or-threonine in the context of specific flanking amino acids (Eglen andSingh, 2003). There are over 400 kinases in humans and their specificityfor phosphorylation sites vary widely. Thus, different antibodies areneeded for assaying diverse kinases or profiling acceptor substrates.This greatly complicates the incorporation of new kinases into HTS,especially if their substrate specificity is not well defined. It alsocreates analysis problems in comparing data among kinases with differentsubstrate selectivities, because the output of the assay depends on theparticular antibody(Ab)-phosphopeptide pair used. Although efforts todevelop generic phospho-serine antibodies and identify more generickinase substrates continue (Sills, M. A., et al., J Biomol Screen, 2002,7:191-214), research in this direction has not been very successful todate.

[0011] A number of alternative approaches have been developed tocircumvent the problem of context-specific Ab-phosphopeptideinteractions, including use of metal complexes to bind phosphopeptides(Scott, J. E. and Carpenter, J. W., Anal Biochem, 2003, 316:82-91) andthe use of modified ATP analogs that allow covalent tagging ofphosphopeptide products (Allison Miller-Wing, E. G., Barbara Armstrong,Lindsey Yeats, Ram Bhatt, Frank Gonzales, and Steven Gessert., SBS 9thAnnual Conference and Exhibition, Portland, Oreg., 2003). Chemicalphosphate binding reagents suffer from background binding to nucleotidephosphates, requiring the use of very low, non-physiological levels ofATP and limiting assay flexibility. Modified nucleotides do not providea generic format because the ability to use the ATP analogs as donorsvaries among kinases, requiring the development of a number of differentanalogs. Also competition of inhibitors with the modified nucleotides atthe kinase ATP binding site—the most frequent site for kinase inhibitorbinding—does not reflect the physiological situation. Differences inprotease sensitivity caused by peptide phosphorylation have also beenexploited in developing fluorescence based kinase assays (Kupcho, K., etal., Anal Biochem, 2003, 317:210-7), but these assays are not trulyhomogenous; i.e. they require the post-reaction addition of developingprotease reagents. In addition, the applicability of this method islimited to peptides where kinase and protease specificity overlap.

[0012] There are also a few methods that are dependent on interaction ofreaction products with specific multiwell plate chemistries, but theseare not truly homogenous in that they require post reaction reagentadditions and/or processing. Also the requirement for specializedinstrumentation for processing and/or detection does not fit with theopen architecture desired by most pharmaceutical HTS platforms.

[0013] Furthermore, microfluidics-based kinase assays that rely onelectrophoretic separation of reaction products have been developed(Xue, Q., et al., Electrophoresis, 2001, 22:4000-7). In these assays,phosphorylated peptide products are electrophoretically separated fromthe non-phosphorylated acceptor substrates, thus eliminating the needfor specialized detection reagents. However, in practice, the kinaseassays are often run in multiwell plates and then the products aretransferred to microfluidic devices for separation—a cumbersome processfor an HTS format.

[0014] In summary, the non-generic nature of the current group transferassays is resulting in significant expense and delays for drug discoverybecause of the need to develop assays for individual enzymes or smallsubgroups within a family. Also, because many of the current assays arebased on modification and detection of specific tagged acceptors, thereis limited ability for testing different acceptor substrates. Often thetagged acceptor substrates used are different from the substrates thatare phosphorylated in vivo, thus the physiological relevance of theassay is questionable. In addition, a major concern in thepharmaceutical industry is that because of the non-generic nature of thecurrent assays, investigators are sometimes forced to use differentmethods for different kinases. However, studies have shown that thereare significant differences in the pharmaceutical targets identifiedusing different assays methods (Sills, M. A., et al., J Biomol Screen,2002, 7:191-214), which is a significant problem for profiling inhibitorselectivity across several kinases. These shortcomings of the existingHTS assay methods for group transfer reactions are hampering the rapidanalysis of important enzyme families in pharmaceutical drug discoveryprograms.

[0015] Other existing approaches for assaying group transfer reactionshave been to enable screening of diverse chemicals as substrates forgroup transfer reactions by detecting the donor molecule product,because it is the same regardless of the acceptor being modified.Detection of the donor product has been thought to provide the basis ofa generic assay method, ADP is always a product of a kinase reaction andphosphoadenosine-phosphate (PAP) is always the product of asulfotransferase reaction. Detection of these products, however, iscomplicated because the cleaved mono- and di-nucleotides cannot bedifferentiated from the activated donor molecules based on absorbance orfluorescence properties because for example ADP has the samefluorescence and absorbance properties as ATP. Separation of the donorproduct from the donor can be effected using chromatographic methods,such as thin layer chromatography or high pressure liquidchromatography, but incorporation of these methods into an HTS format iscumbersome.

[0016] To circumvent this difficulty, detection of the donor product hasbeen achieved by using additional enzymes to generate a detectableproduct from the primary reaction product—the cleaved mono- or di-nucleotide; this is known as an enzyme coupled reaction. For instance,enzymes and other small molecules can be used for ADP-dependentgeneration of NADPH, which is detected by absorbance or fluorescence at340 nm (Walters, W. P. and Namchuk, M., Nat Rev Drug Discov, 2003,2:259-66). An enzyme coupled reaction has also been developed for UGTs,another type of group transfer enzyme (Mulder, G. J. and van Doorn, A.B., Biochem J, 1975, 151:131-40). However, the optical interference ofdrug compounds with absorbance assays, especially in the ultra violet,is a widely recognized problem with this approach. Another shortcomingof this approach is that all of the enzymes used to couple the detectionare subject to potential inhibition from the chemicals being screened.

[0017] Another generic approach is to monitor ATP consumption usingLuciferase as a reporter to detect protein kinase activity. An exampleof this method was disclosed by Crouch et al., in U.S. Pat. No.6,599,711. Their method entailed determining the activity of a proteinkinase to be tested by adding a substrate capable of beingphosphorylated by the protein kinase to a solution having ATP and aprotein kinase to be tested, and another solution having ATP in theabsence of the kinase to be tested. The concentration or the rate oftime change of ATP and/or ADP was then measured using bioluminescence.However this assay is not optimal because it relies on small decreasesin a high initial signal. The need to keep ATP concentrations low tominimize background results in nonlinear reaction kinetics if assayconditions are not carefully controlled. In a related method,competition binding assays using fluorescent ATP analogs have also beendeveloped, but these do not give a measure of enzyme catalytic activity,thus are of limited utility.

[0018] The use of enzymes that catalyze group transfer reactions intoHTS assays has been hampered by the lack of universal, homogenous assaymethods that are not subject to interference from molecules inpharmaceutical drug libraries. Accordingly, it would generally bedesirable to provide universal methods for assaying enzymes involved ingroup transfer reactions that are well suited for HTS drug discovery.

BRIEF SUMMARY OF THE INVENTION

[0019] To enable facile incorporation of important group transferenzymes into HTS assays, applicants have developed assays based onhomogenous immunodetection of the donor product. The general equationfor the group transfer reaction is:donor-X+acceptor→donor-product+acceptor-X, wherein the donor-product isdetected by the general detection reaction: firstcomplex+donor-product→second complex+displaced detectable tag. Becausethe donor product is the same for all enzymes that catalyze a given typeof group transfer reaction, the same detection reagents can be used forall the members within a family of group transfer enzymes and with anyacceptor substrate. The assay products can be detected using homogenousfluorescence or chemiluminescence methods which are not subject tosignificant interference or background signal from molecules inpharmaceutical drug libraries.

[0020] Thus, the present invention is summarized as methods andcomponents thereof for detecting the activity of and screening acceptorsubstrates, inhibitors, or activators of enzymes catalyzing grouptransfer reactions to facilitate the development of more selective andtherapeutic drugs. This is accomplished through a highly selectiveantibody used to bind the donor product of the group transfer reaction.Antibody-antigen interactions can be detected in a number of ways thathave already been described by others. The detection mode applicantshave used to put the method into practice is a competitive fluorescencepolarization immunoassay (FPIA), because it is well suited forpharmaceutical HTS assays. With this detection mode, enzymaticallygenerated donor product displaces a fluorescent derivative of the donorproduct, called a tracer, from an antibody resulting in a decrease intracer fluorescence polarization. The key reagents for the assay are anantibody that binds with high selectivity to the donor product, and notto the uncleaved donor molecule, and a tracer—a fluorescent derivativeof the donor product that retains its structure sufficiently to bind theantibody. The invention provides a novel assay for detecting andquantifying activity for enzymes that catalyze group transfer reactionsusing diverse substrates. The invention also provides a method ofscreening for substrates, inhibitors, or activators of the grouptransfer reactions.

[0021] In one aspect, the invention provides a method of detecting adonor-product of a group transfer reaction, the method includingreacting an activated form of a donor with an acceptor in the presenceof a catalytically active enzyme; forming the donor-product and anacceptor-X; contacting the donor-product with a first complex comprisinga detectable tag capable of producing an observable; competitivelydisplacing the detectable tag of the first complex by the donor productto generate a second complex and a displaced detectable tag; anddetecting a change in the observable produced by the detectable tag inthe first complex and the displaced detectable tag.

[0022] In another aspect, the invention provides an antibody producedagainst a donor product of a group transfer reaction, wherein theantibody has the ability to preferentially distinguish between adonor-product and a donor in the presence of a high donor concentration.

[0023] In yet another aspect, the invention provides a homogeneouscompetitive binding assay for a donor product of a group transferreaction, the assay includes combining the donor-product with a tracerand a macromolecule to provide a mixture, the macromolecule beingspecific for the donor product, the tracer comprising the donor-productconjugated to a fluorophore, the tracer being able to bind to themacromolecule to produce a detectable change in fluorescencepolarization; measuring the fluorescence polarization of the mixture toobtain a measured fluorescence polarization; and comparing the measuredfluorescence polarization with a characterized fluorescence polarizationvalue, the characterized fluorescence polarization value correspondingto a known donor-product concentration.

[0024] In yet another aspect, the invention provides assay kits forcharacterizing a donor-product from a group transfer reaction, the assaykit including a macromolecule and a tracer, each in an amount suitablefor at least one homogeneous fluorescence polarization assay fordonor-product, wherein the macromolecule is a an antibody or aninactivated enzyme.

[0025] Other advantages and a fuller appreciation of specificadaptations, compositional variations, and physical attributes will begained upon an examination of the following detailed description of thevarious embodiments, taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0026]FIG. 1 illustrates a fluorescence polarization immunoassay (FPIA)reaction for the SULT reaction product, PAP.

[0027]FIG. 2 illustrates use of FPIA to detect and quantify UDPformation, the donor product of the UGT reaction.

[0028]FIG. 3 illustrates use of FPIA to detect and quantify ADPformation, the donor product of the kinase reaction.

[0029]FIG. 4 describes a strategy for iterative co-development ofreagents for SULT1E1 FPIA: anti-PAP antibody and fluorescently labeledPAP detectable tag.

[0030] FIGS. 5A-B show titration or competitive displacement curves foruridine nucleotides using a polyclonal antibody raised against UDP/UTPand a commercially available tracer molecule (Alexa-UTP).

[0031]FIG. 6 shows the synthesis of PAP antigens.

[0032] FIGS. 7A-C show components of PAP tracer synthesis.

[0033] FIGS. 8A-C show representative final tracer structures.

[0034]FIG. 9 shows binding isotherms for anti-PAP antibodies andPAP-fluorescein tracers.

[0035]FIG. 10 shows competitive displacement of two different tracersfrom Ab 3642 by PAP and PAPS.

[0036] FIGS. 11A-B show the competition curves with two Anti-PAPantibody/Tracer combinations.

[0037]FIG. 12 shows the effect of PAPS concentration on detection ofenzymatically generated PAP.

[0038] FIGS. 13A-F show a continuous FPIA-based detection of SULTactivity with diverse substrates.

[0039]FIG. 14 shows a comparison of SULT1E1 acceptor substrate profilesdetermined using the FPIA-based assay and the ³⁵S-PAPS radioassay.

[0040]FIG. 15 shows inhibition of SULT1E1 by 2,6 Dichloro-4-nitrophenol(DCNP) measured with the FPIA-based assay.

[0041] Before an embodiment of the invention is explained in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description. The invention iscapable of other embodiments and of being practiced or being carried outin a variety of ways. Also, it is to be understood that the phraseologyand terminology used herein is for the purpose of description and shouldnot be regarded as limiting in any way.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention broadly relates to novel assay methods fordetecting and quantifying the donor-product or the catalytic activitiesgenerating the donor-product from group transfer enzymes using diversesubstrates. The invention also provides the antibodies specific to thedonor product and assay kits for practicing the invention in ahigh-throughput screening format. The general equation for the grouptransfer reaction includes a donor-X+acceptor→donor-product+acceptor-X,wherein the donor-product is detected by the general detection reaction:first complex+donor-product→second complex+displaced detectable tag.Thus, in accordance with the invention, a highly selective antibody isused to bind the donor product of the group transfer reaction, and thisbinding event is detected, using an immunoassay, such as for example acompetitive binding FPIA which is well suited for pharmaceutical HTSassays. By using this detection mode, enzymatically generated donorproduct displaces a tracer, from an antibody resulting in a decrease intracer fluorescence polarization. The key reagents for the assay areantibody that binds with high selectivity to the donor product, and notto the uncleaved donor molecule, and a tracer—a fluorescent derivativeof the donor product that retains its structure sufficiently to bind theantibody.

[0043] In particular the invention provides a universal assay method inthat a single set of detection reagents can be used for all of theenzymes in a given family of group transfer enzymes and all acceptorsubstrates for that family. Because of its universal nature, theinvention will accelerate the incorporation of SULTs, protein kinasesand other group transfer enzymes into HTS screening. For example, thereare eleven known SULT isoforms, using the method of the presentinvention, all eleven of the SULT isoforms may be screened for theirability to sulfate diverse compounds in the same experiment using thesame detection reagents, protocol and instrumentation. This is animportant capability because enzymes that catalyze xenobioticconjugation (e.g., SULTs and UGTs) have very broad acceptor substratespecificity. With respect to kinases, there is even more diversitywithin the enzyme family. There are over 400 protein kinases in humans,and there is great diversity in their acceptors substrate specificitysuch that either physiological protein acceptor substrates as well asshort peptides may be used. As such, a number of protein kinases may beused in the assay of the invention using their diverse acceptorsubstrates and screened for inhibitors using the same detectionreagents, protocol and instrumentation. For these reasons and othersprovided below, the FPIA based donor product detection assays of theinvention for group transfer reactions such as SULT, UGT and kinases,among others are very well suited for automated HTS applications.

[0044] Accordingly, the present invention will now be described indetail with respect to such endeavors; however, those skilled in the artwill appreciate that such a description of the invention is meant to beexemplary only and should not be viewed as being limiting on the fullscope thereof.

Definitions Certain Terms Used Herein are Intended to have the FollowingGeneral Definitions

[0045] The term “group transfer reaction” as used herein refers to thegeneral reaction:

donor-X+acceptor donor→product+acceptor-X.

[0046] Representative group transfer reactions are shown as follows:

[0047] Kinase reaction: ATP+acceptor→ADP+acceptor-PO₄;

[0048] UGT reaction: UDP-glucuronicacid+acceptor→UDP+acceptor-glucuronic acid;

[0049] SULT reaction: acceptor-XH+3′-phosphoadenosine5′-phosphosulfate→acceptor-SO₄+3′-phosphoadenosine 5′-phosphate+H⁺;

[0050] Methyltransferase reaction:s-adenosylmethionine+acceptor→acceptor-CH₃+s-adenosylhomocysteine; and

[0051] Acetyl transferase reaction: acetylCoenzymeA+acceptor→acceptor-COCH₃+CoenzymeA.

[0052] Group transfer reactions in-part such as sulfation by SULTs,phosphorylation by kinases and UDP-glucuronidation by UGTs are suitablyapplicable to the methods of the invention because one can isolateantibody/detectable tag pairs for the donor products, which are PAP, ADPand UDP, respectively. Also group transfer reactions are generallyinvolved in a number of biological processes, such as hormonebiosynthesis and function; enzyme regulation and function; andxenobiotic metabolism.

[0053] The term “universal assay” and “generic assay” are usedinterchangeably to refer to a method whereby all members of the grouptransfer reaction enzyme family and all of their acceptor substrates canbe detected with the same assay reagents.

[0054] The term “covalent adduct” refers to the moiety that istransferred from the donor molecule to the acceptor in a group transferreaction; sulfonate, phosphate, and glucuronic acid respectively forSULTs, kinases, and UGTs.

[0055] The term “donor-product” as used herein refers to the product ofa group transfer reaction that is the fragment of the donor moleculethat is generated when the covalent adduct is transferred to acceptor.Often it is a nucleotide (naturally occurring or synthetic) such as aPAP, UDP or ADP; or a non-nucleotide such as a s-adenosylhomocysteine,nicotinamide or a CoenzymeA. The donor-product is detected by a generalreaction including a first complex+donor-product→secondcomplex+displaced detectable tag.

[0056] The term “tracer” as used herein refers to a displaced detectablederivative or tag of a donor product that retains its structuresufficiently to bind to a specific antibody.

[0057] The term “donor” as used herein refers to a substrate for anenzyme catalyzing a group transfer reaction that carries the activatedcovalent adduct. Examples of suitable donors include not onlynucleotides, but also s-adenosyl methionine and acetyl-CoA, amongothers.

[0058] The term “donor-X ” is another term for donor molecule in which Xrepresents the covalent adduct.

[0059] The term “acceptor” as used herein refers to a substrate for anenzyme catalyzing a group transfer reaction to which the covalent adductbecomes covalently attached, wherein the substrate is a polypeptide, aprotein, a nucleic acid, a carbohydrate, a lipid or a small moleculesubstrate such as a steroid or an amino acid.

[0060] The term “acceptor-X” as used herein refers to a reaction productin which X is the covalently bound covalent adduct; wherein the covalentadduct includes at least one of a phosphate, a sulfate, a carbohydrate,a naturally occurring amino acid, a synthetically derived amino acid, amethyl, an acetyl, or a glutathione moiety, and a combination thereof.The covalent adduct is capable of altering either the function, thestability, or both the function and the stability of the acceptorsubstrate.

[0061] The term “catalytically active enzyme” as used herein refers toat least one of a sulfotransferase, a kinase, aUDP-glucuronosyltransferase, a methyl transferase, an acetyltransferase, a glutathione transferase, or a ADP-ribosyltransferase.

[0062] The term “catalytic activity” as used herein refers to a chemicalcatalytic activity, an enzymatic activity, or a combination thereof.

[0063] The term “first complex” as used herein refers to a complexhaving a macromolecule (i.e., an antibody or an inactivated enzyme) anda detectable tag.

[0064] The term “second complex ” as used herein refers to amacromolecule and the donor product wherein the detectable tag iscompetitively displaced by the donor-product.

[0065] The term “observable” as used herein refers to detectable changein fluorescence, fluorescence intensity, fluorescence lifetime,fluorescence polarization, fluorescence resonance energy transfer (FRET)or chemiluminescence of the second complex or the displaced detectabletag and a combination thereof to obtain a measured observable. Themeasured observable is compared with a characterized observable, whereinthe characterized observable corresponds to the first complex.

[0066] The term “detectable tag ” as used herein refers to a fluorescentor chemiluminescent tracer, which is conjugated to a donor product.Fluorescence is the preferred mode of detection for the invention. Asuitable detectable tag may be produced by conjugating for example, achemiluminescent tag or a fluorophore tag, to the donor product moleculein such a way that it does not interfere significantly with antibodybinding (i.e., most likely attached via the adenine portion of thenucleotide). Fluorophores applicable to the methods of the presentinvention include but are not limited to fluorescein, rhodamine, BODIPY,Texas Red, and derivative thereof known in the art. Rhodamine conjugatesand other red conjugates may be synthesized and optimized as detectabletags because their higher wavelength emission is less subject tointerference from autofluorescence than the green of fluorescein.

[0067] Chemiluminescent tags applicable to the invention include LumigenTMA-6 and Lumigen PS-3 (Lumigen, Inc., Southfield, Mich.) which haveadequate chemiluminescence quantum yield. These reagents possess aneasily measured signal by virtue of an efficient chemiluminescentreaction with a predictable time course of light emission. Furthermore,chemiluminescent tags contribute little or no native backgroundchemiluminescence. Also, measurement of light intensity is relativelysimple, requiring only a photomultiplier or photodiode and theassociated electronics to convert and record signals. In addition,chemiluminescent signals can be generated in an immunoassay using enzymefragment complementation methods, where the detectable tag would be thedonor product conjugated to fragment A of a reporter enzyme, and itsdisplacement from antibody by the donor product generated in the grouptransfer reaction would allow it to associate with fragment B of theenzyme resulting in formation of a catalytically active reporter enzymecapable of acting on a chemiluminescent substrate. This method has beendescribed using β-galactosidase as a reporter enzyme in U.S. Pat. No.4,708,929.

[0068] The term “immunoassay” as used herein may refer to a number ofassay methods wherein the product is detected by an antibody such as forexample a homogenous assay, homogeneous fluorescence immunoassay, ahomogeneous fluorescence intensity immunoassay, a homogeneousfluorescence lifetime immunoassay, a homogeneous fluorescencepolarization immunoassay (FPIA), a homogeneous fluorescence resonanceenergy transfer (FRET) immunoassay or a homogenous chemiluminescentimmunoassay, or a non-homogenous assay such as enzyme-linked immunoassay(ELISA) and a combination thereof.

[0069] The term “fluorescence polarization immunoassay” or “FPIA” asused herein refers to an immunoassay for detecting the products of grouptransfer reactions. Fluorescence polarization (FP) is used to studymolecular interactions by monitoring changes in the apparent size offluorescently-labeled or inherently fluorescent molecules (Checovich, W.J., et al., Nature, 1995, 375:254-6; Owicki, J. C., J Biomol Screen,2000, 5:297-306). When a small fluorescent molecule (tracer) is excitedwith plane polarized light, the emitted light is largely depolarizedbecause the molecule rotates rapidly in solution during the fluorescenceevent (the time between excitation and emission). However, if the traceris bound to a much larger receptor, thereby increasing its effectivemolecular volume, its rotation is slowed sufficiently to emit light inthe same plane in which it was excited. The bound and free states of thefluorescent molecule each have an intrinsic polarization value, a highvalue for the bound state and a low value for the free state. In apopulation of molecules, the measured polarization is a weighted averageof the two values, thus providing a direct measure of the fraction ofthe tracer molecule that is bound. Polarization values are expressed asmillipolarization units (mP), with 500 mP being the maximum theoreticalvalue.

[0070] In practice, small molecules like fluorescein have polarizationvalues of approximately 20 mP, and when bound by an antibody theirpolarization increases by 100 to 400 mP. In a competitive FPIA, afluorescent tracer may be displaced from binding to antibody by thedonor product, as shown in FIG. 1. The signal is proportional to thedifference in the bound versus free tracer fractions, thus both thedynamic range and the sensitivity of the assay are dependent upon theaffinity of the antibody for the tracer and the donor product. Toestablish a suitable dynamic range for an FPIA, approximately 70-80% ofthe tracer must be bound to antibody in the absence of competitor.

[0071] For example, FIG. 1 shows a schematic of a competitive FPIA forthe SULT reaction product PAP in which the PAP produced from the SULTreaction competes with the tracer (fluorescently tagged PAP), forbinding to anti-PAP antibody. In this format, the starting polarizationof the tracer is high because it is almost all bound to antibody, and itdecreases as the reaction proceeds and the tracer is displaced from theantibody. The amount of PAP produced in a SULT reaction can bequantified by using the following equation:${\log \quad\left\lbrack {{PAP}\quad {product}} \right\rbrack} = {\log \quad \frac{\left( {{highest}\quad {polarization}\text{-}{mP}_{observed}} \right)}{\left. {{mP}_{observed}\text{-}{lowest}\quad {polarization}} \right)}}$

[0072] In the enzymatic assay encompassed by the present invention, thePAP for SULTs or ADP for kinases is produced in stoichiometric amountswith the sulfated product or phosphorylated peptide, respectively. Thusthe use of a standard curve for PAP or ADP among other donor productswill allow a direct measure of enzyme turnover.

[0073] The term “high throughput screening” or “HTS” as used hereinrefers to the testing of many thousands of molecules (or test compounds)for their effects on the function of a protein. In the case of grouptransfer reaction enzymes many molecules may be tested for effects ontheir catalytic activity. HTS methods are known in the art and they aregenerally performed in multiwell plates with automated liquid handlingand detection equipment; however it is envisioned that the methods ofthe invention may be practiced on a microarray or in a microfluidicsystem.

[0074] The term “library” or “drug library” as used herein refers to aplurality of chemical molecules (test compound), a plurality of nucleicacids, a plurality of peptides, or a plurality of proteins, and acombination thereof. Wherein the screening is performed by ahigh-throughput screening technique, wherein the technique utilizes amultiwell plate or a microfluidic system.

[0075] The terms “binding molecule” or “macromolecule” as used hereinrefers to an antibody or an inactivated enzyme.

[0076] The term “antibody” as used herein refers to a monoclonal, apolyclonal or recombinant antibody. The antibody is produced against adonor-product of a group transfer reaction and is able to preferentiallydistinguish between a donor-product and a donor in the presence of ahigh donor concentration. The antibody also exhibits specificity towardsat least one of a phosphate portion of a nucleotide, (i.e., an abilityto distinguish between a 5′-phosphate, a 5′-phosphosulfate, a5′-diphosphate and a 5′-triphosphate). For example, in the SULTreaction, the antibody of the present invention differentiates with highstringency between PAP and PAPS. The donor-product molecule for the SULTreaction, PAPS, differs only by the addition of a sulfate group linkedto the 5′ terminal phosphate. This may seem like a relatively smallstructural difference, but the demonstrated ability of antibodies todiscriminate between molecules that differ by a single phosphate—whichis very similar in size and structure to a sulfate group—provides animportant feature of the present invention. For example, an antibody maybe raised against the ribosyl phosphate portion of the molecule.Furthermore, the FPIA-based SULT assay method of the present inventionsuitably requires an antibody that specifically binds the reactionproduct PAP in the presence of excess PAPS.

[0077] In practicing the invention with respect to kinases, antibodiesthat specifically recognize ADP and not ATP may be generated in animalsor by in vitro recombinant methods using ADP conjugated to a carrierprotein in such a way that the phospho-ribosyl portion of the moleculeis exposed, but the adenine portion is largely hidden fromimmunoreactivity. In generating antibodies, it may be helpful to use anonhydrolyzable analog of ADP, such as one containing a methylene orsulfur group bridging the alpha and beta phosphates in order to preventhydrolysis of the immobilized hapten by nucleotidases or phosphatases inthe immunized animals. Alternatively, with respect to methyltransferasesand acetyltransferases, antibodies that specifically recognize smallmolecules other than nucleotides, respectively s-adenosyl homocysteineand Coenzyme A are used.

[0078] The term “inactivated enzyme” as used herein refers to a bindingmolecule that may bind to the donor product. In the case of a kinase,the ADP is the donor product and an inactivated nucleoside diphosphate(NDP) kinase may be the binding molecule. It is known that in manyenzymes, separate domains are involved in binding and in catalysis. Aselective destruction of the catalytic activity with preservation of thebinding properties by genetic engineering, allosteric inhibition, orother chemical means such as taking out cofactors, heme groups, etc. mayenable enzymes, particularly multi-subunit enzymes to function asspecific carriers for their substrates that are no longer able tochemically modify these substrates.

[0079] The term “antibody-detectable tag pair” as used herein refers toan anti-donor product antibody and detectable tag (fluorescent-donorproduct) molecule that allow detection of donor product produced in agroup transfer reaction. A suitable dissociation constant for binding ofthe antibody/detectable tag pair is 1×10⁻⁷M or lower, resulting in aminimal fluorescence polarization shift of 100 mP relative to theunbound detectable tag, and with minimal cross reactivity to the donormolecule at reaction concentrations. The optimal antibody-detectable tagpair may be identified by testing a number of different antibodiesgenerated using different sites of attachment to the donor molecule,different linkers to the carrier protein, and including somenon-hydrolyzable analogs for interaction with a set of detectable tagsgenerated by varying the chemistry of donor molecule attachment to afluorophore. The changes in fluorescence or chemiluminescence of thedetectable tag upon interaction with an antibody that could have afluorophore for example attached t it; may be used as a measure of theirinteraction.

[0080] The term “linker” as used herein refers to spacer arm structures.There are short linkers (i.e., carbamoyl and aminoethyl groups) thatwill sterically minimize the accessibility of the adenine ring andlonger six carbon linkers that will allow more flexible presentation ofthe antigen. The linker molecule affects detectable tag characteristicsin a number of important ways that impact both its antigenic andfluorescence properties. There is generally a balance that must bestruck between separating the antigen from the fluorophore enough toallow unhindered interaction with antibody without creating too muchfreedom of motion for the fluorophore. The former result in loweredaffinity antibody binding and in quenching of the fluorophore, whereasthe latter reduces the polarization shift upon antibody binding, therebyreducing the dynamic range of the assay. Different linkers can beinterchanged using relatively simple chemistry, thus the linker isvaried in a number of ways in efforts to optimize the antigenic andfluorescence properties of the detectable tag. Furthermore, approachesmay be employed similar to those described for PAP conjugation of anantibody involving heterobifunctional linkers to first introduce spacerarms and/or aromatic substituents onto PAP, followed by reaction withreactive fluorescein derivatives. This approach greatly expands therange of possible linker structures.

Methods and Materials

[0081] The following experimental protocols of the invention are notlimited to the particular methodology, protocols, antibodies, enzymes,detectable tags, among other reagents described, as these may varydepending on the group transfer reaction. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention.

[0082] Development of Assay Reagents

[0083] In developing a successful HTS assay format for the invention theaffinity and specificity of assay reagent interaction, and the resultantchanges in detectable tag fluorescence properties ultimately define theoverall performance of the assay, including sensitivity, dynamic range,and signal to noise ratios. As such, to optimize this interaction,applicants have provided an iterative co-development approach as shownin FIG. 4 to most efficiently achieve detection, quantitation andscreening of group transfer reaction products.

[0084] In particular, FIG. 4 describes a strategy for iterativeco-development of reagents for a SULT donor product FPIA: anti-PAPantibody and fluorescently labeled PAP detectable tag. FIG. 4 providesthat to produce diverse antigens for immunization of rabbits, PAP may beconjugated to carrier protein (BSA or KLH) via attachment through theC2, C8 and N6 amino group of adenine using linker molecules of varyinglengths as described below. The same PAP-linker-NH₂ intermediates usedfor antibody conjugation may be used to synthesize fluorescein-PAPconjugates to test as detectable tags. The resulting matrix ofantibodies and detectable tags may be tested for binding using FPassays, and the pairs that exhibit high affinity binding and maximal FPshifts may be used as the basis for further optimization of tracercharacteristics. Optimized Antibody-detectable tag pairs may be testedfor detection of PAP production in sulfation reactions with SULT1E1 oranother SULT isoform.

[0085] Also, it is envisioned that several PAP antigens and detectabletags may be synthesized by conjugating the nucleotide to carrier proteinand fluorescein, while retaining an overall structural bias thatmaximizes antibody recognition of ribosyl-phosphate moieties of the PAPmolecule and minimize crossreactivity with PAPS. Antibodies generatedfrom the PAP antigens may be tested for interaction with the detectabletags to identify the combinations that exhibit optimal binding andfluorescence properties of the novel assay encompassed by the presentinvention.

[0086] Generation of High Affinity Antibodies

[0087] In accordance with methods of the present invention, one skilledin the art may readily prepare a variety of specific antibodies (i.e.,polyclonal, monoclonal or recombinant) against antigenic nucleotidessuch as the ribosyl-phosphate portion of PAP or other chemical moleculessuch as Coenzyme A.

[0088] In general polyclonal antibodies against a range of PAP antigensare suitably generated in rabbits. The injection of animals andcollection of serum may be performed according to the followingprotocol. Three rabbits may be immunized with each of the antigens thatmay be developed. The yields of antiserum from a single rabbit (˜100 ml)are suitable for many thousand to millions of FPIA assays depending onthe titer, affinity, and the multivalent nature of polyclonal-antigeninteractions resulting in very high affinity binding. As such, for thesereasons polyclonals are frequently used for FPIAs (Nasir, M. S. andJolley, M. E., Comb Chem High Throughput Screen, 1999, 2:177-90).

[0089] Also, encompassed within the scope of the present invention aremonoclonal antibodies which may be produced according to the methodsprovided in previously published protocols (Harlow, E. L., D., 1999),which are fully incorporated herein by reference. Also, recombinantsingle chain antibodies, may be generated using the in vitrocombinatorial evolution and display methods previously published(Schaffitzel, C., et al., J Immunol Methods, 1999, 231:119-35;Breitling, F., Dubel, S., 1999). Examples of the preparation ofrecombinant antibodies are described in U.S. Pat. Nos.: 5,693,780;5,658,570; 5,876,961 (fully incorporated by reference herein).

[0090] In order to help elicit an immune response, bovine serum albumin(BSA) and keyhole limpet hemocyanin (KLH) may be used as carrierproteins for conjugation of the PAP antigen. KLH antigens generallyelicit a stronger immune response in mammals, but also tend to be lesssoluble than BSA conjugates (Harlow, E. L., D., 1999). PAP may beattached to both carrier proteins via the C2, C8 and N6 amino group ofadenine as shown in FIGS. 7-8. In accordance with the scope of thepresent invention, development of the FPIA-based SULT assay methodrequires an antibody that specifically binds the reaction product PAP inthe presence of excess PAPS. More suitably, the antibody is capable ofrecognizing the ribosyl phosphate portion of the PAP molecule, and todifferentiate between the 5′-phosphate of PAP and the 5′-phosphosulfateof PAPS.

[0091] Conjugation Methods

[0092] It is known in the art that conjugation to adenine will minimizeantibody cross reactivity with PAPS that could occur via binding to theadenine moiety (Goujon, L., et al., J Immunol Methods, 1998, 218:19-30;Horton, J. K., et al., J Immunol Methods, 1992, 155:31-40; Bredehorst,R., et al., Biochim Biophys Acta, 1981, 652:16-28), and the site ofhapten conjugation also can affect the affinity of the resultingantibodies (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8). Thehapten conjugation chemistries used are well described single or twostep synthetic approaches that have been used extensively forconjugating adenine nucleotides (Brodelius, P., et al., Eur J Biochem,1974, 47:81-9; Lindberg, M. and Mosbach, K., EurJ Biochem, 1975,53:481-6; Camaioni, E., et al., J Med Chem, 1998, 41:183-90). All of thestarting materials are commercially available, and it is understood thatthe reactions may proceed in good yield. These conjugation methods aredescribed below in further detail. In accordance with the methods of thepresent invention, it is envisioned that the present invention mayencompass but may not be limited by the following hapten conjugationchemistries. Attachment to N6 amino group

[0093] The carbamoyl linkage: N6-carboxymethyl-PAP may be generated byalkylation of PAP with iodoacetic acid to form the 1-carboxymethylanalog, which rearranges in base to form the N6-carboxymethyl analog(Lindberg, M. and Mosbach, K., Eur J Biochem, 1975, 53:481-6). TheN6-carboxymethyl-PAP may be conjugated to carrier protein usingcarbodiimide coupling enhanced with N-hydroxysulfosuccinimide. A similarapproach could be used to generate high specificity 5′-AMP antibodies asdescribed in previously published protocols (Bredehorst, R., et al.,Biochim Biophys Acta, 1981, 652:16-28).

[0094] The C6 linkage: N6-aminohexyl-PAP (Sigma/Aldrich, St. Louis, Mo.)may be directly conjugated to carrier proteins by a) converting proteinamino groups (mostly lysines) to reactive thiols using N-succinimidylS-acetylthioacetate (Pierce, Rockford, Ill.) and b) conjugating theterminal amine group on the PAP linker to the protein thiols usingheterobifunctional amine-thiol reactive linkers (Pierce, Rockford,Ill.). It is noteworthy that the N6 amino group of adenosine is forpractical purposes unreactive; for instance it reacts very poorly withsuccinimidyl esters.

[0095] Attachment to Adenosine C2

[0096] The 2-chloroadenosine may be phosphorylated by reaction withphosphorous oxychloride followed by phosphorous trichloride to produce3′,5′-bisphospho-chloroadenosine. Subsequent reaction withethylenediamine and diaminohexane and purification by anion exchangechromatography may be used to produce the 2-aminoethyl- and6-aminohexyl-analogs of PAP at the 2 position of adenine (Brodelius, P.,et al., Eur J Biochem, 1974, 47:81-9). It is envisioned that thesemolecules may react with carrier proteins using heterobifunctionallinkers as described above.

[0097] Attachment to Adenosine C8

[0098] The C2 and C6 linkages to the 8 position of PAP may be generatedusing the same strategy described for the C2 position, except thestarting material may be 8-bromoadenosine (Sigma/Aldrich). Also,8-azido-PAP (ICN, Costa Mesa, Calif.) may be directly bound to carrierproteins by UV irradiation.

[0099] Thin layer chromatography (TLC) may be used to monitor changes inthe adenine ring absorption spectrum. Anion exchange (Dowex resin) andphase separations may be used for antigen purification. To assure highantigen purity, the final PAP-linker molecules to be used forconjugation to carrier proteins may be purified by HPLC and theiridentity verified by mass spectra and NMR analysis. TheN6-carboxymethyl-PAP may be conjugated to methylated BSA, since use ofthe methylated carrier protein has been reported to prevent carbodiimidereaction with the ribose ring (Bredehorst, R., et al., Biochim BiophysActa, 1981, 652:16-28).

[0100] The other PAP analogs all tend to have a reactive amine at theend of the linker, which may be used for conjugation to BSA and KLHusing more specific amine and thiol chemistry, thereby avoiding reactionwith the ribose. The conversion of protein amines to thiols and linkingvia heterobifunctional linkers may result in a higher density of antigenconjugation than direct conjugation to cysteines because there are manymore reactive lysines than cysteines in the carrier proteins. PAPdensity on the carrier protein may be determined by spectrophotometricanalysis for the adenine ring and conjugation chemistries may beadjusted if needed to obtain a high density (>10 PAP per proteinmolecule) and thus insure a robust immune response. It is envisionedthat about 8-10 different antigens may be synthesized in all, some withvery short linkers such as the carbamoyl and aminoethyl groups that willsterically minimize the accessibility of the adenine ring and also withlonger six carbon linkers that will allow more flexible presentation ofthe antigen.

[0101] Synthesis and Purification of Fluorescent PAP Molecules

[0102] In general, an FPIA detectable tag molecule can be divided intothree different structural components: the antigen, the fluorophore, andthe linker used to join them; an additional key structural variable isthe site of attachment of the linker to the antigen. It is understood byone skilled in the art that suitable antigens, fluorophores and linkersare not limited to what is specifically described herein. However, in asuitable aspect of this invention the fluorophore, fluorescein, may beused to conjugate it to the same sites on adenosine used for antigenconjugation to carrier protein. The same PAP-linker molecules that areused for conjugation to carrier proteins may be used for conjugation toseveral amine-reactive fluorescein derivatives. The use of a range ofreactive fluoresceins may introduce additional structural diversity inthe linker region. For instance, fluorescein succinimidyl esters areavailable with different length spacer arms, and DTAF(4,6-Dichlorotriazinylaminofluorescein) contains a bulky planarsubstituent adjacent to the site of attachment. Likewise, theintroduction of an aromatic ring to AMP near the site of carrier proteinconjugation (N6-benzyl AMP) enhances its affinity for antibody by40-fold relative to unmodified AMP (Bredehorst, R., et al., BiochimBiophys Acta, 1981, 652:16-28). Briefly, various PAP-linker moleculeswith amino termini may be reacted with amine-reactive rhodamine, TexasRed, and other red fluorophors and the resulting PAP- fluorophorsconjugates may be tested for antibody binding affinity and changes influorescence polarization.

[0103] Purification of PAP-conjugates can be performed by thin layerchromatography (TLC). TLC is used to separate reaction products becauseit allows several separations to be run in parallel, permitsidentification of fluorescent products by visualization and, providessufficient quantities of fluoresceinated conjugates for thousands ofbinding assays in a single chromatography run. Thus for pilot studies onfluorescent detectable tag molecules, TLC is superior to alternativeseparation methods like HPLC. Upon completion of the chromatography andelution of the PAP-fluorophore conjugates, the conjugates may beanalyzed for intensity, basal polarization (absence of antibody),stability, binding to anti-PAP antibodies, and competition by unlabeledPAP and PAPS, as described below.

[0104] Parameters Affecting the Antibody-Detectable Tag Interaction

[0105] In order to create the most sensitive assay method for detecting,quantifying, and screening products of group transfer methods, theparameters affecting the affinity of the antibody-detectable taginteractions is carefully monitored. This is important, for example, inthe case of SULT1E1 where the potent substrate inhibition exhibited bysome acceptor substrates, may limit the amount of product that can begenerated. The affinity required for the novel SULT assay encompassed bythe present invention is estimated by considering the limiting case ofthe high affinity, inhibitory substrate, estradiol. In general, theK_(i) for estradiol is 80 nM and the K_(m) is about 20 nM (Zhang, H., etal., J Biol Chem, 1998, 273:10888-92), so using 50 nM estradiol in theassay and adjusting enzyme and reaction time so that 10% of thesubstrate is consumed—resulting in formation of 5 nM PAP—is a goodcompromise that may allow measurement of initial velocity withoutcausing significant inhibition.

[0106] To allow detection of 5 nM PAP the required affinity for antibodybinding to fluorescent-PAP (and unconjugated PAP) may be approximately25 nM. Most antibody-antigen interactions with either polyclonal ormonoclonal antibodies have a K_(d) between 1 nM and 1 μM (Harlow, E. L.,D., 1999). For instance, the anti-phosphotyrosine antibodies used inFPIA-based kinase assays, exhibit subnanomolar affinity forphosphorylated peptides and their fluorescent conjugates (Parker, G. J.,et al., J Biomol Screen, 2000, 5:77-88), and the anti-AMP polyclonalantibody described above bound to the nucleotide with an affinity of 100nM (Bredehorst, R., et al., Biochim Biophys Acta, 1981, 652:16-28). Itis difficult to predict how the structure of the antigen will affect theaffinity of the resulting antibodies, but the carrier protein and modeof conjugation both can have a profound effect (Crabbe, P., et al., JAgric Food Chem, 2000, 48:3633-8; Signorini, N., et al., Chem ResToxicol, 1998, 11:1169-75; Oda, M. and Azuma, T., Mol Immunol, 2000,37:1111-22). Accordingly, antibodies may be generated using PAPconjugated to two different proteins via different sites on PAP andusing different length linker regions.

[0107] Another factor affecting the sensitivity of PAP detection is poorsubstrates that are sulfated very slowly even at high concentrations. Inthese cases, the amount of SULT1E1 enzyme required becomes a limitingfactor because of cost considerations. Using a substrate that is turnedover at 1% the rate of the V_(max) with estradiol, it may require 30 ngof SULT1E1 to generate 5 nM PAP—a suitable detection limit in about 30minutes. This is well within the acceptable range, as it may mean that a100 mg batch of enzyme may support more than three million reactions.

[0108] Furthermore, the difference in polarization of the probe in thefree and bound states defines the total “spread” or dynamic range forthe assay. A change of less than 50 mP may be sufficient forsemi-quantitative detection of SULT1E1 activity, but a change of 100 mPor greater may provide both much greater flexibility in designing theassay format, and more quantitative kinetic information. Accordingly,polarization is proportional to molecular volume, and the change ineffective volume upon binding of an antibody (150 kDa) to a smallmolecular weight fluorophore may be expected to cause an increase of atleast 300 mP. However, there are other factors that can affect theobserved polarization of both the free and bound detectable tag, suchas, the fluorophore is attached to PAP by a flexible spacer arm. Thus,their maximum polarization is a function of the extent to which antibodybinding decreases the mobility of the spacer and fluorophore; detectabletag “propeller effects” can lead to relatively low polarization valueseven for protein-bound fluorophores.

[0109] An additional consideration is whether there may be changes inthe intensity of the fluorophores upon binding to receptor, includingquenching from amino acid functional groups with overlapping spectra.Polarization is independent of fluorescence intensity (Owicki, J. C., JBiomol Screen, 2000, 5:297-306); however, because it is a ratio ofintensity measurements taken in two planes, significant quenching mayaffect the antibody detectable tag interaction and result in decreasedassay sensitivity. The changes in the fluorescence properties of adetectable tag when it becomes bound by antibody are a function of bothinteracting molecules and are difficult to predict. Thus, it isenvisioned that different antibodies and detectable tag molecules willbe tested in a matrix fashion. Further optimization of the detectabletag structure or the assay buffer to limit mobility of the bounddetectable tag or to reduce quenching is also envisioned.

[0110] Identification of the Optimal Antibody/Detectable Tag Pair(s)

[0111] In order to identify optimal antibody/detectable tag pairs, adiverse set of fluorescein derivatives and linkage chemistries aregenerally evaluated. The synthesis and purification methods describedabove result in the isolation of several fluorescent products perreaction, so that at least a hundred fluorescent conjugates may beevaluated, requiring thousands of individual FP assays. The homogenousnature of FP assays and the availability of multiwell instruments makesthe screening efforts very rapid; the rate-limiting steps are usuallysynthesis and purification, not testing for Antibody-detectable tagbinding properties. The antibody/detectable tag testing may be carriedout in a Tecan Ultra multiwell FP reader in Tris or phosphate buffer inthe presence of a low concentration of carrier protein (0.01% BovineGamma Globulin) to prevent non-specific interactions. The initial screeninvolves looking for the following desirable properties: lowpolarization in the absence of antibody (<100 mP); high affinity bindingto the anti-PAP antibodies (K_(d)<100 nM); maximal difference inpolarization between the bound and free states (Δ mP >100 mP); minimalfluorescence quenching upon binding antibody; selectivity for PAP (i.e.,competitive displacement by PAP and not by PAPS or other adeninenucleotides); rapid association/dissociation kinetics; and lack ofinteraction with SULT1E1 or other assay components.

[0112] For initial antibody binding assays, each of the purifieddetectable tags may be quantified by measuring fluorescence intensitywith the assumption that the intensity of the conjugated fluorescein isnot quenched. This is not always the case, but those with significantquenching may be eliminated through the binding analysis because theyappear to have low binding affinity. Antibody/detectable tag pairs withpromising affinity (K_(d)<100 nM) and fluorescence properties may betested for the specificity of binding by competition with unlabeled PAPand PAPS and other nucleotides. Those Antibody/detectable tag pairs withthe most desirable FPIA properties may be examined in more detail toassess whether any changes in extrinsic conditions such as the additionof salt or detergent may improve their sensitivity or dynamic range.Additional detectable tags may be optionally synthesized and testedbased on the structure-activity relationships observed and thepredictions of computer-generated structural models.

Applicability of the Invention

[0113] The methods of the invention, as described in the examples belowcan be used as a measure of enzymatic activity in group transferreactions. The methods are of particular importance in thepharmaceutical industry since they enable the analysis of group transferenzymes in high throughput screening laboratories, e.g. foridentification of drugs that can act as enzymatic modulators, especiallyinhibitors, and for determining how potential drug molecules aremetabolized.

[0114] There is an increasing number of group transfer enzymes that arebeing targeted for the development of new therapeutics. Kinases arecurrently of highest interest, but SULTs, UGTs, methyltransferases,acetyltransferases, and ADP-ribosyltransferases are also being targeted.As this list increases it will become impractical and extremelyexpensive to use specific tests for each enzyme, or for small subgroupsof enzymes that modify the same acceptor substrate. The assays and kitsof the present invention allow for the detection of all of the enzymesin a given group transfer enzyme family and provide a common end pointdetection system for all of them. This allows for greater ease of use,particularly in high throughput screening laboratories, where robots canbe set up with all the detection reagents and the various group transferenzymes and potential substrates or inhibitors of interest can be testedin a matrix fashion.

[0115] In addition, the involvement of group transfer reactions in theconjugative metabolism of drugs and other xenobiotics is an importantdeterminant in the level of their pharmacodynamics, pharmacokinetics andside effects. In this regard, the assays and kits of the presentinvention allow for the screening of potential drug molecules withSULTs, UGTs, methyltransferases and acetyltransferases in an HTS format.There are many different xenobiotic conjugating isoforms, and because ofthe generic nature of the method, the kits of the present inventionwould greatly simplify their testing in a matrix fashion.

[0116] Thus, the general methods of the invention may be applied to avariety of different group transfer-related enzymatic processes, such assteroid hormone biosynthesis and function, xenobiotic metabolism, enzymereceptor regulation, and signal transduction in an effort to contributeto an integrated drug discovery approach discussed below.

[0117] Sulfation in Drug Discovery

[0118] Sulfation is a ubiquitous covalent modification used to regulatethe levels and activities of endogenous hormones and xenobiotics. Inhumans it is catalyzed by a family of eleven different sulfotransferaseenzymes (SULTs), each with different, but overlapping substratespecificity and tissue distribution(Strott, C. A., Endocr Rev, 2002,23:703-32). Endogenous substrates for sulfation include many importantsignaling molecules, such as steroid hormones, catecholamines, andthyroid hormones; xenobiotics that are sulfated include drugs,promutagens and environmental endocrine disruptors (Strott, C. A.,Endocr Rev, 2002, 23:703-32; Glatt, H., et al., Mutat Res, 2001,482:27-40; Coughtrie, M. W. and Johnston, L. E., Drug Metab Dispos,2001, 29:522-8). Sulfation is reversible and can change the activity ofsignaling molecules, often by altering their affinity for receptorproteins, thus it serves as an on-off switch for receptor ligands, muchas phosphorylation serves that role for proteins. Moreover, the role ofsulfation in xenobiotic metabolism is intertwined with its involvementin the regulation of hormonal signaling and cell homeostasis. Forinstance, sulfation regulates the activity of endogenous ligands forspecific neurotransmitter receptors, nuclear receptors, and proteinkinases that are drug targets for depression, breast cancer, andcardiovascular disease (Plassart-Schiess, E. and Baulieu, E. E., BrainRes Brain Res Rev, 2001, 37:133-40; Strott, C. A., Endocr Rev, 1996,17:670-97; Kuroki, T., et al., Mutat Res, 2000, 462:189-95). Developmentof more selective therapies for these disorders by pharmaceuticalcompanies is currently hampered by a lack of molecular assays andpurified SULT isoforms for high throughput screening (HTS) of potentialSULT substrates and inhibitors.

[0119] There are two major classes of SULTs in humans that differ insolubility, size, subcellular distribution, and share less than 20%sequence homology. The membrane bound enzymes are located in the golgiapparatus and sulfonate large endogenous molecules such as heparan,glycosaminoglycans, and protein tyrosines (Strott, C. A., Endocr Rev,2002, 23:703-32). The cytosolic sulfotransferases, or SULTs sulfonatesmall molecular weight xenobiotics and hormones.

[0120] There are eleven known cytosolic SULT isoforms, which differ intheir tissue distribution and specificity. The nomenclature used isbased on homology at the amino acid level: SULTs within the same family,indicated by a number, share at least 45% amino acid identity, and thosewithin the same subfamily, designated by a letter, share at least 60%identity. The SULTs can be differentiated to some degree based onsubstrate specificity, though their substrate profiles overlapextensively, even between enzymes in the different families.

[0121] Structural studies have revealed that there is flexibility in thesubstrate binding sites and that ligand binding elicits the transitionto a more ordered structure, which may contribute to the broad substratespecificity of the SULTs (Bidwell, L. M., et al., J Mol Biol, 1999,293:521-30). For this reason, it is not likely that computationalapproaches such as molecular docking may be applicable for identifyingSULT substrates and inhibitors; this increases the value of the HTSassays encompassed by the present invention.

[0122] It is envisioned that the assays of the invention may be used inan HTS format to provide for example, SULT metabolism information suchas whether the compound of interest interacts with one or more SULTisoforms, whether the compound is a substrate or inhibitor, and thekinetic parameters (IC₅₀, K_(m), V_(max)) for the interaction. The HTSassay encompassed by the present invention provides all of thisinformation by screening in two different modes: a) direct measurementof test compound turnover; and b) measurement of compound inhibition ofprobe substrate sulfation. In the direct turnover mode, compounds couldbe screened vs. a panel of SULT isoforms to profile compound sulfationby individual SULT isozymes. After isozyme identification, more detailedkinetic studies with the appropriate SULT isozyme could be used topredict in vivo clearance rates (Obach, R. S., et al., J Pharmacol ExpTher, 1997, 283:46-58), reducing the failure rate of compounds inclinical studies (Greco, G. N., E; Martin, Y C, 1998, 219-245). SULTinhibitors could be identified by using a known substrate—e.g. estradiolfor SULT1E1—and screening test compounds for inhibition. Isozymespecific kinetic and inhibition data will further improve drug discoveryby providing knowledge of metabolism by specific SULT alerting the drugdiscovery team to potential drug-drug interactions and pharmacogeneticissues. The contribution of genetic differences in SULTs to varyingresponses to therapeutics is an active area of investigation (Thomae, B.A., et al., Pharmacogenomics J, 2002, 2:48-56).

[0123] Identification of the SULT responsible for the metabolism of adrug will aid in judicious selection of the in vitro assays or animalmodels used for preclinical assessment of possible drug-druginteractions and toxicology testing, thereby reducing inappropriate orunnecessary experimental animals use. Metabolism data can be used as acomponent of rational drug design and lead optimization. A betterunderstanding of the structure-activity relationships that definesubstrate specificity for the various SULT isozymes may provide a basisfor structural modifications of primary compounds to change theirmetabolism profile (Greco, G. N., E; Martin, Y C, 1998, 219-245).Accordingly, the methods of the present invention will enable a betterunderstanding of the structure-activity relationships that definesubstrate specificity for the various SULT isozymes.

[0124] Integrated Approach to Drug Discovery

[0125] The ability of the assays of the invention to be used as HTSassays enables a rational, integrated approach to drug development fortherapeutic areas where for example, sulfation is a component of therelevant signal transduction biology. Some specific areas where the HTSmethods of the invention may be used include for example steroid hormonebased therapies. Sulfation, in accordance with the present invention,encompasses involvement in estrogen level regulation in mammary tumors,as well as androgen levels in prostate tumors. Furthermore, cortisolsulfation, though not well understood, inactivates the hormone forbinding to the glucocorticoid receptor. High throughput biochemicalassays for steroid ligand-receptor binding and the resultant binding oftranscriptional coregulator proteins are already commercially availableand being used by pharma HTS groups to find more selective steroidhormone modulators (Parker, G. J., et al., J Biomol Screen, 2000,5:77-88; Spencer, T. A., et al., J Med Chem, 2001, 44:886-97). As such,the availability of robust HTS assays for steroid sulfation may providean important addition to the arsenal of molecular tools available topharma groups focused on steroid signal transduction. For instance, theinhibition of SULT1E1 by compounds targeting the ER could causedeleterious effects by elevating estrogen levels in tumor cells.Molecules with SULT1E1 inhibitory properties could be culled or modifiedbased on studies using the assay methods of the present invention.

[0126] The modulation of neurosteroids is being investigated as a novelpharmacological approach to controlling neural excitatory balance(Malayev, A., et al., Br J Pharmacol, 2002, 135:901-9; Maurice, T., etal., Brain Res Brain Res Rev, 2001, 37:116-32; Park-Chung, M., et al.,Brain Res, 1999, 830:72-87). The methods encompassed by the presentinvention may suitably accelerate these efforts by allowing facilescreening of endogenous and synthetic neurosteroids forsulfoconjugation, offering insight into the fundamental biology as wellas providing a tool for lead molecule identification and optimization.The need for better molecular tools is accentuated by the fact thatthere is already a sizeable over the counter market for DHEA as an“anti-aging” dietary supplement purported to alleviate age relatedsenility and memory loss (Salek, F. S., et al., J Clin Pharmacol, 2002,42).

[0127] Furthermore, it is envisioned that the methods of the presentinvention may suitably identify drug targets with respect to cholesterolsulfate in the regulation of cholesterol efflux, platelet aggregationand skin development in treatments for cardiovascular disease andperhaps some forms of skin cancer. In this instance, asulfotransferase—most likely SULT2B1b—could become the drug target, andmolecules that selectively inhibit this isoform may need to beidentified. The availability of a full panel of the human SULTs and arobust HTS assay method of the present invention may be valuable to suchan effort.

[0128] Glucuronidation in Drug Discovery

[0129] Drug metabolism problems such as production of toxic metabolitesand unfavorable pharmacokinetics cause almost half of all drug candidatefailures during clinical trials (Obach, R. S., et al., J Pharmacol ExpTher, 1997, 283:46-58). All of the major pharmaceutical companies haverecognized the need to consider pharmacokinetic and pharmacogenomicconsequences early in the drug discovery process resulting in animmediate need for high throughput in vitro methods for assessing drugmetabolism. Aside from P450-dependent oxidation, glucuronidation isperhaps the most important route of hepatic drug metabolism. A broadspectrum of drugs are eliminated or activated by glucuronidationincluding non-steroidal anti-inflammatories, opioids, antihistamines,antipsychotics and antidepressants (Meech, R. and Mackenzie, P. I., ClinExp Pharmacol Physiol, 1997, 24:907-15; Radominska-Pandya, A., et al.,Drug Metab Rev, 1999, 31:817-99). Despite their importance, the broadand overlapping substrate specificity of the hepaticUDP-glucuronosyltransferases (UGTs) that catalyze glucuronidationremains poorly understood because of a lack of flexible in vitro assaymethods. The primary reasons for this is that the catalytic assays usedrequire separation of reactants from products, which involvessubstrate-specific chromatographic steps and thus is not practical in anHTS format.

[0130] Two UGT families (e.g., UGT1 and UGT2) have been identified inhumans; although the members of these families are less than 50%identical in primary amino acid sequence, they exhibit significantoverlap in substrate specificity. The members of the UGT1 family thatare expressed in human liver, where the majority of xenobioticmetabolism takes place, include UGT 1A1, 1A3, 1A4, 1A6, and 1A9.Although the UGT2 family has not been studied as extensively, it isknown that UGT2B4, 2B7, 2B10, 2B11 and 2B15 are expressed in the liver.Mutations in UGTs are known to have deleterious effects, includinghyperbilirubinaemia which occurs with a frequency of 5-12% (Weber, W.,1997) and can lead to neurotoxicity and in severe cases, death. As isthe case for other drug metabolizing enzymes such as P450s,interindividual differences in UGT expression levels have been observedand linked to differences in drug responses (Iyer, L., et al., J ClinInvest, 1998, 101:847-54). For instance, low expression of UGT1A1, as inpatients with Gilbert's syndrome, has been associated with the toxicityof Irinotecan, a promising anticancer agent (Wasserman, E., et al., AnnOncol, 1997, 8:1049-51). In addition, UGT upregulation in tumor tissueshas been identified as a possible cause of anticancer drug resistance(Franklin, T. J., et al., Cancer Res, 1996,56:984-7; Takahashi, T., etal., Jpn J Cancer Res, 1997, 88:1211-7).

[0131] All of the known UGTs exhibit broad substrate specificity, with asingle isozyme catalyzing glucuronidation of a broad range ofstructurally unrelated compounds; not surprisingly there also is a greatdeal of overlap in the specificities of UGT isozymes (Radominska-Pandya,A., et al., Drug Metab Rev, 1999, 31:817-99). With regards tobiotransformation of endogenous molecules, UGT 1A1 is clearly thepredominant isoform involved in glucuronidation of the tetrapyrrole,bilirubin, resulting in its excretion. Beyond this, it is difficult tomake generalizations regarding specificity because of the lack ofsystematic studies with most of the recently identified isoforms.Numerous endogenous steroids have been identified as aglycones for mostof the hepatic isoforms include including 1A1, 1A3, 1A4, 2B4, 2B7, and2B15. Lipids and bile acids serve as substrates for 2B4 and 2B7, andrecently retinoids have been identified as substrates for some isoformsfrom both families. The structural diversity of known xenobioticaglycones is very broad; it includes many drugs and drug like moleculesincluding tertiary amines such as imipramine, non-steroidalanti-inflammatories (NSAIDs) such as acetominophen and naproxen, opioidssuch as morphine and codeine, and carboxylic acid containing drugs suchas clofibric acid.

[0132] In the short term, pharmaceutical companies have an immediateneed for better methods to determine whether their potential drugcandidates will be glucuronidated in vivo, and if so by which UGTisoform. And in the long term, developing the ability to predict themetabolism of drugs by glucuronidation will require a systematic effortto fully define the “chemical space” recognized by each of the keyhepatic UGTs. The proposed Phase II studies will generate the moleculartools required for this effort, including HTS assay methods that can beused to rapidly screen large numbers of diverse chemicals for bindingand metabolism by isolated UGT isoforms.

[0133] Pharmaceutical research and development is time consuming,expensive, and inefficient, resulting either in higher costs or loweravailability of new therapies for the U.S. health care consumer.Currently, development of a new drug in the USA requires ten to fifteenyears and a total R&D outlay of $400 to $750 million. While clinicaltrials are the most expensive phase of development, typically accountingfor 30-50% of the total R&D cost, only 10% of all drug candidates testedin clinical trials ultimately are commercialized (Obach, R. S., et al.,J Pharmacol Exp Ther, 1997, 283:46-58). Moreover, an analysis of thereasons for drug candidate attrition during clinical trials confirmsthat some of the key determinants of the success or failure of acompound are a function of its metabolism, including rate of clearance(pharmacokinetics), potential for interference with the metabolism ofother drugs, and potential for generating toxic metabolites. Thecombination of poor pharmacokinetics—usually excessively rapidclearance—and toxicity cause over 50% of all clinical failures. Gaininga better understanding of how potential drug candidates are metabolizedearly in the discovery phase would improve the success rate inpreclinical and clinical studies resulting in more efficient drugdevelopment, and increased economy and availability of therapies.

[0134] It is envisioned that the immunoassay (i.e., FPIA-based donorproduct assay) for UGTs will be used in a manner very similar to thatdescribed for SULTs for determining whether potential drug candidatesinteract with any of the known UGT isoforms. Using the method of theinvention, it will be possible to determine whether compound of interestinteracts with a one or more UGT isoforms, and if so, whether it is asubstrate or inhibitor. Also one can identify the kinetic parameters(IC₅₀, K_(m), V_(max)) for the interaction between the compound ofinterest and enzymatic isoform. It should be noted that recombinantforms of the many of the UGT isoforms are already available (Invitrogen,Becton-Dickinson). The information obtained with these HTS assays can beused in the following ways such as after isozyme identification, moredetailed kinetic studies with the appropriate UGT isozyme can be used topredict in vivo clearance rates, reducing the number of compounds thatfail in clinical studies due to poor pharmacokinetics. Also, theknowledge of metabolism by a specific UGT alerts the drug discovery teamto potential pharmacogenetic problems, since genetic differences in UGTlevels are recognized as an important factor in varying responses totherapeutics. Furthermore, the identification of the UGT responsible forthe metabolism of a drug will aid injudicious selection of the in vitroassays or animal models used for preclinical assessment of possibledrug-drug interactions and toxicology testing, thereby reducinginappropriate or unnecessary use of animals for experiments. Also,metabolism data can be used as a component of rational drug design. Abetter understanding of the structure-activity relationships that definesubstrate specificity for the various UGT isozymes would provide a basisfor structural modifications of primary compounds to change theirmetabolism profile. Also, the testing of glucuronidated compounds canlead to the discovery of valuable prodrugs that are inactive untilmetabolized in the body into an active form.

[0135] Protein Kinases in Drug Discovery

[0136] There are more than 400 distinct kinases encoded in the humangenome; elucidating their role in disease and identifying selectiveinhibitors is a major pharma initiative. Kinase malfunction has beenlinked to all of the most important therapeutic areas, including cancer,cardiovascular diseases, inflammation, neurodegenerative diseases, andmetabolic disorders. Moreover, clinical validation of kinases as drugtargets has recently been shown in the cases of Herceptin and Gleevec,which inhibit aberrant tyrosine kinases that contribute to breast cancerand leukemia, respectively. High throughput screening (HTS)—the paralleltesting of many thousands of compounds for interaction with a drugtarget—has become the dominant mode of drug discovery. The total marketfor HTS assay reagents in 2002 was $474M, and approximately 20% ofscreening was done on protein kinases. Despite the high level ofinterest, pharma efforts to incorporate kinases into HTS programs arehampered by shortcomings with the assay methods. The most commonly usedHTS kinase assays rely on fluorescence-based immunodetection of aphosphorylated peptide reaction product, which varies with the substratespecificity of individual kinases. Time consuming reagent development isthus required for each kinase, or group of related kinases, andcomparison of results between assays is problematic. To overcome thistechnical hurdle the invention provides for a FPIA for detection ofadenosine diphosphate (ADP), a product of all kinase reactions. Thisassay will accelerate efforts to define kinase substrate specificity andto identify novel inhibitors by providing a universal catalytic assaythat can be used with any kinase and any acceptor substrate.

[0137] Protein Kinases are a large, diverse family with a key role insignal transduction. Protein kinases, which catalyze the transfer of theterminal phosphate group from ATP or GTP to serine, threonine ortyrosine residues of acceptor proteins, are one of the largest proteinfamilies in the human genome. In the broadest senses, they can bedivided into serine/threonine or tyrosine kinases and soluble enzymes ortransmembrane receptors. In the most recent and comprehensive genomicanalysis, 428 human kinases were identified that comprise eightdifferent homology groups, which also reflect differences in substratespecificity, structure/localization and/or mode of regulation (Hanks, S.K., Genome Biol, 2003, 4:111). For instance, there are 84 members of theTyrosine Kinase group, which includes both transmembrane growth factorreceptors such as EGFR and PDGFR and soluble enzymes such as the Srckinases, 61 members of the cyclic nucleotide dependent group, ser/thrkinases which includes the lipid dependent kinases—the PKC isoforms, and45 members of the “STE” group, which includes the components of themitogenic MAP kinase signaling pathway.

[0138] Kinases are ubiquitous regulators of intracellular signaltransduction pathways, and as such have come under intense focus bypharmaceutical companies searching for more selective therapies for abroad range of diseases and disorders; they are second only to G-proteincoupled receptors in terms of pharma prioritization (Cohen, P., Nat RevDrug Discov, 2002, 1:309-15). Intracellular targets for phosphorylationinclude other kinases, transcription factors, structural proteins suchas actin and tubulin, enzymes involved in DNA replication andtranscription, and protein translation, and metabolic enzymes (Cohen,P., Trends Biochem Sci, 2000, 25:596-601). Phosphorylation can causechanges in protein catalytic activity, specificity, stability,localization and association with other biomolecules. Simultaneousphosphorylation at multiple sites on a protein, with differentfunctional consequences, is common and central to the integration ofsignaling pathways.

[0139] Diversity of Phosphorylation Sites.

[0140] Each kinase may phosphorylate one or more target proteins,sometimes at multiple sites, as well as autophosphorylate within one ormore regulatory domains that control catalytic activity or interactionwith other biomolecules. Defining the functional consequences ofcellular phosphorylation profiles for normal and disease states is amajor proteomics initiative. However, to use this knowledge for decidingwhich kinases to target for drug discovery, their specificity foracceptor substrates must also be delineated. Kinases recognize specificlinear sequences of their target proteins that often occur at betabends. In general, amino acids that flank the phosphorylated residue forthree to five residues on either side define a phosphorylation site. ThePhosphoBase database, which compiles known kinase phosphorylation sites,contains entries for 133 human kinases, less than a third of the totalkinases. Moreover, most, if not all of these specificity profiles areincomplete, as they only show one or two peptides that have beenidentified as substrates for each kinase. Though there is significantoverlap in substrate specificity among related kinases, there is noconsensus sequence that is phosphorylated by a large number of kinases.This situation complicates the incorporation of diverse or novel kinasesinto HTS assays that rely on detection of specific phosphorylatedproducts. TABLE 1 Target Structure: Company (Phase) EGFR Small molecule:GlaxoSmithKline (I), Pfizer (II), WyethAyerst (I), AstraZeneca (III),OSI Pharmaceuticals Monoclonal: Imclone (III), Abgenix (II), Merck (I),Medarex/Merck (I) Her2/Neu Monoclonal: Genentech (Herceptin), Medarex/Novartis (I), Genentech (I) PDGFR/ Small molecule: Novartis (Gleevec)cKit/BCR- Abl Raf Small Molecule: Merck, Onyx/Bayer (II) Antisense: IsisPharmaceuticals (II) MEK Small molecule: Pfizer (II), Promega (I) CDKSmall molecule: Aventis (II), Cyclacel (I), Bristol-Myers Squibb (I) PKCSmall molecule: Novartis (II), GPC Bioteck (II), Eli Lilly (I)Antisense: Isis (III)

[0141] Table 1. Selected clinical trials for development of kinaseinhibitors as anticancer agents (Dancey, J. and Sausville, E. A., NatRev Drug Discov, 2003, 2:296-313). Bolded drugs are approved

[0142] Protein Kinases in Cancer

[0143] The biological rationale for targeting kinases to intervene incancer is far too extensive to attempt an overview here. However, one ofthe dominant themes is the involvement of numerous kinases incontrolling the delicate balance between the rate of cell division (cellcycle progression), cell growth (mass), and programmed cell death(apoptosis) that is perturbed in all cancers. Growth factor receptortyrosine kinases (RTKs) are membrane-spanning proteins that transducepeptide growth factor signals from outside the cell to intracellularpathways that lead to activation of progrowth and anti-apoptotic genes.The majority of the fifty-eight RTKs in humans are dominant oncogenes,meaning that aberrant activation or overexpression causes a malignantcell phenotype. Not surprisingly, tyrosine kinases are beingaggressively pursued as anticancer drug targets and both small moleculeand monoclonal antibody inhibitors—Gleevec and Herceptin,respectively—have been clinically approved. Downstream signaling fromgrowth factor receptors occurs through multiple pathways involving bothser/thr and tyrosine kinases. One of the dominant kinases is the mitogenactivated protein kinase (MAPK) pathway, which includes Raf and MEKkinases; inhibitors of all of these kinases are currently being testedin clinical trials (Table 1) (Dancey, J. and Sausville, E. A., Nat RevDrug Discov, 2003, 2:296-313). Soluble tyrosine kinases, especially the11 oncogenes that comprise the Src family, also transduce mitogenicsignals initiated by RTKs and are being targeted by pharma (Warmuth, M.,et al., Curr Pharm Des, 2003, 9:2043-59). Following mitogenic signalsthrough RTKs that initiate entry into the G1 phase, progression throughthe cell cycle is regulated by sequential activation of phase-specifickinases in association with cyclin proteins. The cyclin dependentkinases represent yet another important group of kinases that pharma ispursuing in the hopes of inhibiting malignant cell proliferation(Table 1) (Elsayed, Y. A. and Sausville, E. A., Oncologist, 2001,6:517-37).

[0144] Kinases as Targets in Other Diseases

[0145] Pharma interest in kinases is most intensely focused on cancer,but extends to all of the key therapeutic areas. Table 2 shows recentreviews describing the biological rationale for pursuing kinases targetsfor a broad range of disorders; note that the targets overlap thosebeing pursued for cancer. Entire companies have formed on the basis oftargeting kinases for drug discovery. These include Sugen(http://www.sugen.com, now owned by Pfizer), Signase(http://www.signase.com/index.htm), and ProQinase(http://www.proginase.com/index.html). TABLE 2 Disease TargetsInflammatory diseases, MAPK (Adams, J. L., et al., Prog Med Chem, 2001,38: 1-60); arthritis MEK 1, 2 (English, J. M. and Cobb, M. H., TrendsPharmacol Sci, 2002, 23: 40-5) Type II diabetes and GSK-3(Eldar-Finkelman, H., Trends Mol Med, 2002, 8: 126-32), PI-complications 3 Kinase (Jiang, G. and Zhang, B. B., Front Biosci, 2002,7: d903-7), PKCβ (Frank, R. N., Am J Ophthalmol, 2002, 133: 693-8), IRTK(Laborde, E. and Manchem, V. P., Curr Med Chem, 2002, 9: 2231-42), PKA(Musi, N. and Goodyear, L. J., Curr Drug Targets Immune Endocr MetabolDisord, 2002, 2: 119-27) Hypertension, PKC, var. isoforms (Malhotra, A.,et al., Mol Cell Biochem, 2001, cardiovascular 225: 97-107), Rho Kinase(Chitaley, K., et al., Curr Hypertens Rep, 2001, 3: 139-44) Neuropathy -CDK5 (Lau, L. F., et al., J Mol Neurosci, 2002, 19: 267-73); MAPKAlzheimer's disease, (Dalrymple, S. A., J Mol Neurosci, 2002, 19:295-9), CKDs (O'Hare, M., stroke et al., Pharmacol Ther, 2002, 93:135-43) Neuropsychiatric PKC α, PKC ε (Chen, G., et al., Bipolar Disord,2000, 2: 217-36) disorders Antimicrobials Histidine Kinases (Matsushita,M. and Janda, K. D., Bioorg Med Chem, 2002, 10: 855-67)

[0146] Table 2. Listing of Links between Protein Kinases and VariousDiseases.

[0147] It is envisioned that the FPIA-based donor product assay will beused to screen drug libraries for inhibitors or activators of proteinkinases. It will also be useful for screening peptides or proteins asacceptor substrates for kinases. In these applications, it will have thesignificant advantages over other methods such as the universal natureof the assay, simplified homogenous assay, no radioactivity, and theability to quantify enzyme turnover.

[0148] Universal Assay Method

[0149] This method will accelerate the incorporation of protein kinasesinto HTS screening programs because it is truly generic: a single set ofdetection reagents can be used for all kinases and all acceptorsubstrates. An important advantage over most of the current assaymethods is the capability to use physiological protein acceptorsubstrates as well as short peptides.

[0150] Homogenous

[0151] This assay is a single addition, mix and read format. This is animportant factor driving decisions on assay selection in an automatedhigh throughput environment (High Tech Business Decisions, M., CA,Commisioned Market Analysis, 2002). In addition, if antibodies withsuitable binding kinetics are isolated, it allows a continuous assayformat that provides more detailed kinetic information than a stop-timeassay.

[0152] Fluorescence Detection

[0153] By employing fluorescent probes, the FPIA format eliminatesradiation handling, disposal and costs. It should be noted that over thelast few years FP has become one of the key HTS assay platforms used bypharma (Owicki, J. C., J Biomol Screen, 2000, 5:297-306). It is expectedthat in 2003 it will be used by pharma in approximately 12% of totalprimary screening assays; this is a doubling from the level of FP usagein 2001 (High Tech Business Decisions, M., CA, Commisioned MarketAnalysis, 2002). FP is a standard mode on several commercial HTS platereaders.

[0154] Quantitative

[0155] In the proposed enzymatic assay, the ADP is produced instoichiometric amounts with the phosphorylated peptide or protein, thusthe use of a standard curve for ADP will allow a direct measure ofenzyme turnover. Though the use of FP for HTS applications is arelatively recent development, the use of FPIAs for quantitativedetection of hormones and metabolites in a diagnostic setting is verywell established (Nasir, M. S. and Jolley, M. E., Comb Chem HighThroughput Screen, 1999, 2:177-90).

EXAMPLES Example 1 Uridine Glucuronide Transfersase Assay

[0156] One embodiment of this application involves detecting the “donorproduct” of the UGT reaction using a competitive fluorescencepolarization immunoassay where the antibody-bound tracer has a highpolarization value which decreases when it is displaced by an analyte,such as UDP (as shown in FIG. 2). The main reagent required for thisassay is the production of an antibody that binds UDP with highselectivity and has negligible binding to the donor, UDP-glucuronic acid(UDPGA). This highly selective antibody is used in combination with acommercially available fluorescent UTP compound to establish the assay.

[0157] The polyclonal antibody produced against UDP required covalentbinding to a carrier protein. UTP was used as the hapten only becausereactive derivatives of the triphosphate, but not the diphosphate, werereadily available that could be used for conjugation. It was reasonedthat the majority of the triphosphate may be hydrolyzed to di- andmono-phosphate in animals. Several different chemistries for linking theUTP to carrier protein were investigated, because the nature of thelinkage can have a profound affect on the resulting antibody specificityand affinity for antigen. Care was taken so that the linker molecule wasattached to the uridine ring rather than the ribose or phosphate, thusmaximizing the immunoreactivity with the portion of the UDP moleculethat may differentiate it from the donor, UDPGA.

[0158] Rabbit antiserum raised against a mixture of UTP and UDPconjugated to BSA and a commercially available tracer molecule, afluorescently labeled UTP compound (Alexa-UTP, Molecular Probes) wasadded to wells of a black multiwell plate (Thermo Labsystems Pt#7605)containing the indicated amounts of uridine nucleotides. Alexa-UTP wasused as a tracer for the FPIA experiments. Fluorescence polarization wasread in a Tecan Ultra plate reader after several hours of equilibration.Reaction conditions were as follows: 50 mM KPO₄ pH 7.4, 150 mM NaCI, 0.1mg/ml BGG, 1 riM ChromaTide Alexa Fluor 488-5-UTP, 1.25 ul rabbit sera,100 μl total volume.

[0159] The experimental results from the UGT reaction are provided inFIGS. 8A-B. FIG. 8 shows titrations of antibody-tracer complex withvarious uridine nucleotides using the first polyclonal antibody raisedagainst UDP/UTP and a commercially available tracer molecule(Alexa-UTP). It is noted that the two graphs differ in the scale of theX-axis and that competition by UDP, the donor product is half maximal atapproximately 10 μM, whereas for UDPGA, the donor, half maximaldisplacement is higher than 1 mM which is at least a 100× difference inselectivity.

[0160] Most relevantly, whereas UDP displaces the tracer at lowmicromolar concentration, there is no detectable crossreactivity withUDPGA at concentrations in excess of 100 μM. The crossreactivity withUTP is not problematic for the proposed assay because it is not present,nor is it produced, in UGT enzyme reactions. The ability of UDP todisplace the tracer at low micromolar concentrations means that thisantibody is suitable for detection of UDP produced in UGT enzymereactions. Furthermore, since, through preliminary binding kineticstudies there have been indications that the displacement of tracer byUDP is very fast, use of antibody for monitoring UGT turnover in realtime; i.e., a continuous assay, is encompassed within the invention.

[0161] Despite their importance, the broad and overlapping substratespecificity of the hepatic UDP UGTs that catalyze glucuronidationremains poorly understood because of a lack of flexible in vitro assaymethods. The primary problem is that the catalytic assays used requireseparation of reactants from products, which involves substrate-specificchromatographic steps and thus is not practical in an HTS format. Byestablishing the concept for measuring the UGT reaction product, UDP ina fluorescent, homogenous format, the applicants have provided thetechnical foundation for solving this problem. Because the assaymeasures a product common to all UGT reactions, it allows a single modeof detection with any UGT isoform and any substrate. In addition, themethod does not require separation of reactants from products, and is asignificant improvement over other related assays because it usesfluorescence detection rather than colorimetric, making it moresensitive and more desirable for pharma HTS platforms, which have becomelargely reliant on fluorescence based detection. Also, it is acontinuous assay method, thus can provide real time kinetic data on UGTenzyme turnover. In addition the antibody-antigen binding reaction isless susceptible to interference from test samples than a conventionalcoupled enzyme reaction that has been used in the past for donor productdetection (Mulder, G. J. and A. B. D. Van Doorn, Biochem J., 1975, 151:p. 131-40). Thus, the novel assay method will enable screening ofdiverse compounds for metabolism by a panel of isolated UGT isozymes,which will greatly enhance preclinical metabolism studies, andpotentially reduce the clinical attrition rate.

[0162] Furthermore, the properties of the antibody produced by theapplicants have broader implications for development of HTS assays forother important classes of enzymes. As shown in FIG. 8, UDP, but not UMPdisplaces the tracer, illustrating that the antibody is capable ofdifferentiating between nucleotides on the basis of a single phosphategroup. Thus, it is encompassed that antibodies against other “donorproducts” such as ADP for protein kinase reactions where the reactionproduct differs from the donor by a single phosphate may be produced.

Example 2 Kinase Assays

[0163] The assay method of the invention relies on FPIA detection of akinase reaction product ADP that is produced in stoichiometric amountswith phosphorylated polypeptide. Similar to the UGT assay describedabove, the components of the FPIA based donor product kinase assayinclude an antibody to the donor product and a tracer comprised of adonor product conjugated with a detectable tag. The antibody is highlyspecific for ADP (i.e., it is capable of recognizing ADP in the presenceof excess ATP and a fluorescent tracer). The antibody and tracer in akinase assay are added to wells of a black multiwell plate atconcentrations optimal for the start of the assay. A suitableconcentration of tracer is 1-2 nM and sufficient antibody is used tocause approximately 75% of the maximal polarization shift for thetracer. The acceptor substrate is added to the wells at the desiredconcentration, generally 2-5-fold higher than the Km value. The acceptorsubstrate can be a peptide or an intact protein. One benefit of theinvention is that any acceptor substrate can be used, whereas otherkinase assay methods require a specific acceptor for detection. A bufferthat is compatible with the kinase to be assayed and the antibody-tracerinteraction, generally a Tris-Cl or phosphate buffer at about a neutralpH is used. With the buffer, the other required components of the assayare added, including ATP at a concentration of 100 μM-5 mM, MgCl₂, andany other agents required for activation or stabilization of the kinase.The kinase enzyme is then added to initiate the reaction and thepolarization values are monitored in a multiwell reader such as theTecan Ultra. As the reaction proceeds, ADP produced in stoichiometricamounts with the phosphorylated peptide or protein displaces the tracerfrom the antibody resulting in decreased polarization values.

[0164] In screening for inhibitors, the compounds to be tested aregenerally dispensed into wells prior to addition of any other assaycomponents, and control wells with no inhibitor added are included forcomparison.

[0165] Each kinase may phosphorylate one or more target proteins,sometimes at multiple sites, as well as autophosphorylate within one ormore regulatory domains that control catalytic activity or interactionwith other biomolecules (Cohen, P., Trends Biochem Sci, 2000,25:596-601). Defining the functional consequences of cellularphosphorylation profiles for normal and disease states is a majorproteomics initiative, and this knowledge can be used for deciding whichkinases to target and their specificity for acceptor substrates in drugdiscovery. Though there is significant overlap in substrate specificityamong related kinases, there is no consensus sequence that isphosphorylated by a large number of kinases. This situation complicatesthe incorporation of diverse or novel kinases into HTS assays that relyon detection of specific phosphorylated products.

Example 3 Sulfotransferase Assays

[0166] Expression and Purification of SULT1E1

[0167] In order to establish a sulfotransferase HTS assay method,SULT1E1, a SULT isoform, was first subcloned into an E. coli expressionvector with a C-terminal 6× histidine tag and the expressed protein waspurified by affinity chromatography and characterized with respect toits physical and enzymatic properties.

[0168] The purified protein migrated close to its calculated molecularweight on SDS-PAGE and more importantly, mass spectral analysis agreedvery closely with predicted molecular weight (36,161 and 36,374 daltons,respectively). Because it was designed to retain a native N-terminus,the purified C-terminal 6×His construct was also subject to N-terminalsequencing; 15 amino acids were sequenced and they were identical withthe human SULT1E1 sequence in GenBank (NP00541 1). Further sequenceverification was obtained through mass spectral analysis and proteinsequencing.

[0169] To serve as a comparison, the enzymatic properties of thepurified SULT1E1 were examined. Estradiol and estrone were used asphysiological substrates and additional positive (α-naphthol) andnegative (dopamine) control compounds tested to assess specificity. Twotypes of radioassays were used for these studies, one using ³⁵S-PAPS(Foldes, A. and Meek, J. L., Biochim Biophys Acta, 1973, 327:365-74) ,and the other using ³H-estradiol (Zhang, H., et al., J Biol Chem, 1998,273:10888-92); both have been used extensively for SULT characterizationand are described below. TABLE 4 Kinetic Parameters w/Estradiol RelativeReaction Rates with Various acceptors V_(max) α- Construct K_(m) (nM)nmol/min/mg V_(max)/K_(m) Estradiol Estrone naphthol DHEA DopaminehSULT1E1- 15 130 8.67 100% 39% 41% 11% 2% 6xHis

[0170] Table 4 shows the enzymatic properties of SULT1E1 fusionproteins. K_(m) and V_(max) determinations were done using ³H-estradiolradioassays in which the radioactive sulfated product is separated fromunreacted ³H-estradiol by organic/aqueous phase extraction and countedin a liquid scintillation counter. Kinetic parameters were calculatedfrom V vs. S curves by nonlinear regression using GraphPad/Prizmsoftware. The relative reaction rates with various acceptors weredetermined using 400 nM acceptor substrate in the ³⁵S-PAPS radioassay,in which the unreacted ³⁵S-PAPS is precipitated as a barium-metalcomplex and the supernatant containing the ³⁵S-labeled sulfoconjugate iscounted in a liquid scintillation counter. Basal reaction conditions inboth cases were 10 mM KPO₄ pH 6.5, 10 mM DTT, 1.5 mM MgCl₂, 10 mM PAPS,0.5 ng SULT1E1, 0.0025 to 15 mM acceptor.

[0171] The V_(max) and estradiol K_(m) values determined for thepurified SULT1E compared favorably with published values for purifiedrecombinant SULT1E1, which are 30-40 nmol/min/mg and 5-15 nM,respectively. The published data on acceptor substrate specificity ismore varied, but the results reflect the general trend that estradioland estrone are very good substrates, α-naphthol is intermediate, andDHEA and dopamine are very poor substrates (see Table 4). Thus all ofthe SULT1E1 constructs applicants expressed showed native substratespecificity and catalytic rates similar to the highest published values.

[0172] Synthesis of Antigens and Generation of Antibodies

[0173] As to the actual development of the fluorescence-based HTS assayfor sulfotransferases, there were four components showing a PAP FPIAthat detected SULT catalysis: synthesis of antigens and generation ofantibodies; synthesis of tracer molecules; testing antibodies andtracers for interaction and specificity; and demonstrating detection ofSULT1E1 activity using optimal antibody-tracer pairs. Tracer synthesisefforts overlapped significantly with antigen synthesis efforts, as thesame reactive PAP derivatives were used for conjugating to carrierprotein and fluorophores.

[0174] Development of the proposed FPIA-based SULT assay method requiresan antibody that specifically binds the product of the SULT reaction,PAP, in the presence of excess PAPS; i.e., an antibody thatdiscriminates on the basis of a single 5′-sulfate group. There is ampleprecedent for antibodies that discriminate between various nucleotidesthat differ only in the number of phosphates, which is similar in sizeand structure to a sulfate group as described above. However, there wasno precedent for generation of antibodies that specifically recognizePAP.

[0175] Small molecules like PAP must be conjugated to a carrier proteinin order to be used as an immunogen. Suitably, an antigen density of10-20 per carrier protein is optimal. As discussed above, the twoelements of our antigen synthesis strategy were a) synthesis and testingof several antigens because the site of attachment to nucleotide andlinker structure can profoundly affect the properties of the resultingantibodies (Crabbe, P., et al., J Agric Food Chem, 2000, 48:3633-8;Signorini, N., et al., Chem Res Toxicol, 1998, 11:1169-75; Oda, M. andAzuma, T., Mol Immunol, 2000, 37:1111-22), and b) conjugation via theadenine ring because this may allow free exposure of the 5-phosphategroup and limit immunoreactivity with the adenine portion of themolecule, resulting in antigens with the desired specificity. None ofthe nitrogens in the adenine ring were reactive enough to conjugatedirectly to protein or via crosslinking reagents, so reactive PAPderivatives were used as a starting point; the only commerciallyavailable reagent was N6-aminohexyl PAP. What initially appeared to bethe most straightforward approach—reacting N6-aminohexyl-PAP withcarrier proteins by carbodiimide coupling or amine-reactivecrosslinkers—generated unacceptably low antigen density, despite asignificant experimental effort.

[0176] To pursue alternative chemistries applicants outsourced thesynthesis of the photoreactive molecules, 2- and 8-azido-PAP andC8-hexylamino-PAP (FIG. 6). FIG. 6 shows the synthesis of PAP antigens.2- and 8-azido-PAP, which were custom synthesized by ALT, Inc.(Lexington, Ky.) were irradiated (254nm) in the presence of BSA;unreacted nucleotides were removed by filtration and dialysis. Finalantigen densities of 7-12 PAP/BSA were obtained as determined byabsorbance of the adenine ring (shifted to 270-280 nm). Rabbits (3 perantigen) were immunized by Lampire Biologicals (Ottsville, Pa.).N6-aminohexyl PAP (Sigma) was conjugated to KLH using glutaraldehyde andinjected into three rabbits by Biosynthesis Corp. (Lewisville, Tex.).Immunization schedules were similar for all antigens and included 3-4injections over a 4-6 week period.

[0177] Antigens from the two photoreactive PAP derivatives were producedand sent out for antibody production; generation of antigen fromN6-aminohexyl PAP using glutaraldehyde crosslinking to KLH andproduction of antibody was contracted out to a separate company (FIG.6). All three antibodies were reported by the contract producers to bindPAP as determined by ELISA (data not shown).

[0178] In addition, applicants synthesized the reactive derivativesN6-carboxymethyl PAP and 2′-O-succinyl-PAP (FIG. 7); antibodies fromthese derivatives have not yet been produced. A detailed description ofAb binding properties using competitive FPIA is provided below followingthe description of tracer synthesis.

[0179] Synthesis of PAP-Fluor Tracers

[0180] An FPIA tracer molecule can be divided into three differentstructural components: the antigen, the fluor, and the linker used tojoin them; an additional key structural variable is the site ofattachment of the linker to the antigen. Because identification of atracer is largely empirical, applicants used a variety of linkers tojoin PAP and fluorescein via different sites on each molecule; in mostcases the final linker region is a composite of the reactive fluoresceinand PAP molecules used.

[0181] The antibody strategy was to conjugate through the adenine moietyin order to generate antibodies that bind specifically to theribosyl-phosphate group of PAP. The same sites are the obvious sites forfluor conjugation as well in order to leave the desired immunoreactiveportion of the tracer molecule free to bind Ab. Also, fluorescein wasthe preferred fluor used for conjugation. Though red-shifted fluors suchas rhodamine are more desirable for HTS applications, development of FPtracers with fluorescein is usually more straightforward because it isless prone to non-specific binding effects and there are numerousactivated derivatives available. As to the linker molecule, it affectstracer characteristics in a number of important ways that impact bothits antigenic and fluorescence properties. There is generally a balancethat must be struck between separating the antigen from the fluor enoughto allow unhindered interaction with antibody without creating too muchfreedom of motion for the fluor. The former can result in loweredaffinity Ab binding and in quenching of the fluor, whereas the latterreduces the polarization shift upon Ab binding, thereby reducing thedynamic range of the assay.

[0182] N6-aminohexyl PAP was the only activated PAP molecule used forimmunogen synthesis that was useful for tracer synthesis; thephotoactivation reactions required to conjugate the two azido-PAPderivatives may be inefficient for joining two small molecules. Toprovide PAP molecules activated at different positions and withdifferent linker regions, applicants outsourced the production ofC8-aminohexyl-PAP and synthesized in house two PAP derivatives withcarboxy-terminal linkers: N6-carboxymethyl-PAP and 2′-O-succinyl-PAP.Though the latter compound is linked through the ribose hydroxyl ratherthan the adenine, this approach has been used to generate highlyspecific antibodies and tracers for cAMP (Horton, J. K., et al., JImmunol Methods, 1992, 155:31-40).

[0183] Below are brief descriptions of the synthesis and purification ofactivated PAP molecules and their conjugation to various activatedfluors; tracer synthesis components are summarized in FIGS. 7 and 8. Ingeneral, PAP molecules with amino-terminal linkers were conjugated tofluors activated with succinimidyl esters (or isocyanate in one case)and PAP molecules with carboxy-terminal linkers were reacted withfluorescein derivates containing free amino groups using carbodiimidecoupling.

[0184]FIG. 7 illustrates structures of components of Tracer Synthesis.FIG. 7 from left to right provides PAP molecules with amino-terminallinkers attached at the C8 and N6 position, amine- and carboxy-reactivefluorescein derivatives, and PAP molecules with carboxy-terminal linkersat the N6 and 2′-OH. The fluorescent PAP conjugates were separated byTLC and tested for binding to anti-PAP antibodies. In accordance to theinvention, sixteen reactions were run containing different combinationsof PAP and fluorescein derivatives and each reaction yielded 1-4fluorescent products that could be resolved by TLC. In all, more than 40tracers have been purified and tested for binding to Ab. N6-aminohexylPAP (Sigma) and all of the reactive fluorescein derivatives (MolecularProbes) are commercially available. 2′-O-succinyl-PAP andN6-carboxymethyl-PAP were synthesized as described below; C8-aminohexylPAP was synthesized by Jena Biosciences (Jena, Germany). FIG. 8 providesrepresentative final tracer structures.

[0185] Preparation of N6-carboxymethyl PAP

[0186] To prepare N6-carboxymethyl PAP, 100 mg PAP was incubated with0.3 g Iodoacetic acid in 1.2 mL aqueous, adjusted to pH 6.5 with LiOH.The reaction proceeded at 30° C. for 5-7 days, periodically adjustingthe pH to 6.5. The resulting 1-carboxymethyl-PAP product wasprecipitated with ethanol and reconstituted in distilled water and thepH was adjusted to 8.5 with LiOH. This reaction was heated at 90° C. for1.5 hours to yield the N6-carboxymethyl PAP. This product was purifiedon a Dowexl-X2 (200-400 mesh) column equilibrated in 0.3 M LiCl, pH2.75. A gradient was applied over 10 column volumes using 0.5M LiCl, pH2.0. N6-carboxymethyl PAP eluted off the column as pure product and wasconfirmed by mass spectral analysis, ˜20% yield.

[0187] Preparation of 2′-O-Succinyl-PAP

[0188] To prepare 2′-O-Succinyl-PAP, 10 mg (0.024 mmole) PAP andsuccinic anhydride (43 mg, 0.426 mmole) were dissolved in distilled H₂Ocontaining 10% triethylamine (v/v, 1 mL) and shaken for 1.5 hours withreaction progress monitored using reverse phase thin layerchromatography (RP-TLC). Upon completion, the reaction was lyophilizedtwice to ensure no residual triethylamine remained. The expectedsuccinate was column purified using Fast Flow Sepharose-Q resin(Pharmacia) equilibrated with 50 mL 250 mM NH₄OAc, and eluted with alinear gradient of 500 mM to 750 mM NH₄OAc (50 mL of each). Fractionscontaining the product were concentrated on a rotovap followed bylyophilization to furnish 8.4 mg or 67% yield as determined byabsorbance measurement. Stock solution was stored at −20° C. for futureuse.

[0189] Preparation of Fluorescein Conjugates with Amino-Activated PAP

[0190] To prepare fluorescein conjugates with amino-activated PAP, 10 μLof a 100 mM solution of N6- or C8-aminohexyl-PAP in distilled H₂O wascombined in a screw cap vial with a molar equivalent of a fluoresceinsuccinimidyl ester, the reaction was brought to a final volume of 200 μLusing anhydrous dimethyl sulfoxide (DMSO), and shaken on a vortexer for24 hours. Reaction progress was followed by RP-TLC using H₂O as thedeveloping media. Reaction products were purified using preparative TLCon normal phase silica gel using 1:1 EtOH/0.5 M NH₄OAc. Fluoresceinlabeled compounds—generally 3-5 produced in each reaction—werevisualized using UV light, scraped from the TLC plates, and extractedfrom the silica gel using 1:1 MeOH/0.5 M NH₄OAc. Individual fractionswere shaken on a vortex for 1 hour wrapped in aluminum foil andcentrifuged at 4000 RPM for 8 minutes. The supernatants were trituratedto a separate labeled vial and the extraction process repeated. Combinedsupernatants were standardized to 50 nM solution for future use andfrozen in amber microfuge tubes at −20° C.

[0191] Preparation of Fluorescein Conjugates with Carboxy-Activated PAP

[0192] To prepare fluorescein conjugates with carboxy-activated PAP, 10ml of a 100 mM solution of N6-carboxymethyl-PAP or 2′-O-succinyl-PAP indistilled H₂O was combined with 50 equivalents of1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) (10 μL of a 1Msolution in anhydrous. DMSO) followed by 75 equivalents ofN-hydroxysuccinimide (15 μL of a 1000 mM solution in anhydrous DMSO),the reaction was brought to an intermediate volume of 180 μL withanhydrous DMSO and shaken for 48 hours. One molar equivalent (20 ml) offluorescein derivative with a free primary amine was then added and thereaction was incubated with vortexing for 24 hr. Reaction progress wasfollowed by RP-TLC using H₂O as the developing media. Upon completion ofthe reaction, fluorescent products were purified by normal phase silicaTLC and eluted as described above, except that 9:1 acetonitrile/2mMNH₄OAc, pH 5.5 was used as the TLC solvent and 8:2 acetonitrile/2 mMNH₄OAc, pH 5.5 was used for elution.

[0193] A total of 16 unique combinations of activated PAP andfluorescein molecules were reacted and from these, more than 40fluorescent products were isolated and tested for binding to antibodies.This approach can be viewed as sort of a “poor man's combinatorialchemistry,” applicants have found it to be very successful fordevelopment of FP tracers in other instances. It is noted that thehomogenous nature of FP assays and the availability of multiwellinstruments makes the screening efforts relatively rapid.

[0194] Characterization of Anti-PAP Antibodies and Tracers

[0195] Analysis of antibody/tracer interaction was done by fluorescencepolarization. To measure fluorescence polarization (described brieflyabove), the emission intensity is measured in both the vertical andhorizontal planes, which are parallel and perpendicular to thevertically polarized excitation light. The emission light definespolarization: P=(horizontal intensity−vertical intensity)/(horizontalintensity+vertical intensity) Polarization values are reported as mP,which is 1000 times the polarization value (30 mP=0.03 P). Fluoresceinhas a polarization of approximately 20 mP at 37° C. in phosphate bufferat pH 7.0. Mathematically, the limiting polarization for fluorescein is500 mP; practically, an anti-fluorescein antibody bound to fluoresceingives a polarization of ˜450 mP. The currently available multiwell FPreaders such as the Tecan Ultra can read polarization values with greatprecision; i.e., detection of 1 nM fluorescein in the free and the boundstate with less than <3 mP standard deviation.

[0196] In a competitive FPIA, a fluorescently labeled antigen, a tracer,is displaced from binding to antibody by the analyte. The signal isproportional to the difference in the bound versus free tracerfractions, thus both the dynamic range and the sensitivity of the assayare dependent upon the affinity of the antibody for the tracer and thecompeting sample molecule. To establish a suitable dynamic range for anFPIA, at least 70-80% of the tracer must be bound to antibody in theabsence of competitor. This normally is achieved by using the tracer ata concentration 2-10 fold below the Kd and the antibody at aconcentration 2-3-fold over the K_(d). The useful concentration range ofthe assay then ranges from several-fold below the K_(d) concentration toabout 20-fold over the K_(d) concentration. For example, if the K_(d)for the tracer/antibody complex is 10 nM, then the range of detectionfor the sample may be from about 2 nM to about 200 nM. If the K_(d) isten fold less (1 nM), the sensitivity of the assay also improves about10-fold.

[0197] Acceptable properties for anti-PAP antibody and tracer in termsof the assay parameters affected are defined as 1) dynamic range: lowtracer polarization in the absence of Ab (<100 mP) and maximaldifference in polarization between the bound and free states (Δ mP>100mP); 2) sensitivity: high affinity binding of tracer to the anti-PAPantibodies (K_(d)<100 nM), displacement by free PAP with a similar IC₅₀,and minimal fluorescence quenching caused by binding; 3) signal/noise:high Ab selectivity; i.e., competitive displacement of tracer by PAP andnot by PAPS or other adenine nucleotides; also lack of tracerinteraction with SULT1E1 or other assay component; and 4) a continuousassay: rapid association/dissociation kinetics.

[0198] Results of Ab-Tracer Interaction Studies

[0199] Initial screens for Ab-tracer binding were done by adding severalconcentrations of each antibody to 1 nm tracer in multiwell plates andmonitoring for increases in tracer polarization. With all of theantibodies, purification of IgG fractions using immobilized Protein A orProtein G was required to remove non-specific binding components. All ofthe data shown is using antibodies purified on Protein G (Pierce Nab™Protein G Spin Chromatography Kit). Tracer/Ab combinations that showedan interaction in the initial screen were analyzed in more detail forcompetitive displacement by PAP and selectivity of PAP over PAPS. Atotal of nine purified antibodies (3 antigens ×3 rabbits) were testedfor binding with 44 different fluorescein-labeled PAP tracers. Allassays were done in black 96-well plates or black low-volume 384-wellplates, which gave essentially identical results, and polarizationvalues were read on a Tecan Ultra with a fluorescein filter set(excitation at 485 nm to emission at 535 nm) (Ex₄₈₅/Em₅₃₅).

[0200] The binding isotherms in FIG. 9 were generated using the threeantibodies and two tracers shown in Table 5 below, and arerepresentative of experiments used to screen antibodies and tracers forbinding. TABLE 5 Representative antibodies and tracers AbbreviationDescription Ab 1781 8-azido-PAP-BSA immunogen Ab 1810 2-azido-PAP-BSAimmunogen Ab 3642 N6-aminohexyl-PAP-KLH immunogen N6-PAP-F8N6-aminohexyl-PAP conjugated to 6-carboxyfluorescein succinimidyl esterC8-PAP-F14 C8-aminohexyl-PAP conjugated to 6-carboxyfluoresceinsuccinimidyl ester

[0201] Also, FIG. 9, provides binding isotherms for anti-PAP antibodiesand PAP-fluorescein tracers. Shown are the N6-PAP-F8 tracer binding toAbs 1781 ◯, 1810 ⋄, and 3642 □, and C8-PAP-F14 tracer with the sameantibodies: 1781 , 1810 ♦, 3642 ▪. Antibodies were serially dilutedtwo-fold in 50 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, 0.1mg/mL BGG, and 1 nM tracer in a total volume of 100 uL in a 96-wellplate or 12 ul in 384-well plates (results in the two plates areidentical) and polarization values were read on the Tecan Ultra afterone hour incubation at room temperature.

[0202] The binding properties of antibodies generated from the sameimmunogen in different animals were similar, but not identical. In thisexperiment, the 3642 antibody bound both tracers significantly tighterthan 1781, and 1810 did not bind either tracer even at the highestconcentration tested. The maximal polarization values for 3642 bindingto N6-PAP-F8 and C8-PAP-F14 were 140 and 300 mP, respectively; unboundtracer polarization was approximately 20 mP, so the shift observed ismore than adequate for use in an FPIA. To test whether binding isspecific, competitive displacement by PAP was assessed.

[0203]FIG. 10 shows PAP and PAPS competition curves for Ab 3642 and thesame two tracers, PAP and PAPS. N6-PAP-6F8 tracer is represented by theopen symbols: ◯(PAP), □ (PAPS). C8-PAP-F14 tracer is represented withthe closed symbols:  (PAP), ▪ (PAPS). PAP or PAPS was serially dilutedtwo-fold in black mutiwell plates containing 50 mM phosphate buffer (pH7.4), 150 mM NaCl, 0.1 mg/mL BGG, 1 nM C8-PAP-F14, and 1.5 uL purifiedAb 3642 in a total volume of 12 μl or 1 nM N6-PAP-F8 and 0.5 uL 3642 Abin a 20 uL volume. Polarization values were read after one hour at roomtemperature.

[0204] The IC₅₀ for PAP with the C8-PAP-F14 tracer is 300 nM, low enoughto allow use of these reagents for monitoring SULT1E1 activity. Notethat a much higher concentration of PAP (and PAPS) is required tocompete off the tighter binding N6-PAP-F8 tracer. This may be because inthis case the tracer has the same linker group as the immunogen used togenerate antibody, and a population of antibody is recognizing thelinker, making the tracer more difficult to displace with free PAP. FIG.10 also shows that PAPS is less effective than PAP at displacing thetracers from Ab 3642.

[0205] Similar results were observed with the 1781 Ab as shown in FIG.11, which includes complete curves for PAP, PAPS and several similarmolecules in competition experiments with the C8-PAP-F14 tracer. FIG. 11shows competition curves with two anti-PAP antibody/tracer combinations.Competitors were serially diluted two-fold in a black 384-wellmicrotiter plate containing 50 mM phosphate buffer (pH 7.4), 150 mMNaCl, 0.1 mg/mL BGG, 1 nM Tracer C8-PAP-F14, and either 1.5 uL Ab 3642(A) or 3 uL Ab 1781 (B) in a total volume of 12 uL. After one hourincubation at room temperature, the polarization values were read on aTecan Ultra (Ex₄₈₅/Em₅₃₅). The mean and standard deviation of duplicatesfor all data sets are shown. The IC₅₀ values for PAP and PAPS with the3641/tracer and 1781/tracer combinations were 0.3 uM PAP, 3.8 uM PAPS;and 0.3 uM PAP, 1.5 uM PAPS, respectively.

[0206] The 1781 and 3642 antibodies exhibited a 5- and 13-foldselectivity for PAP over PAPS respectively, and much higher selectivityfor PAP over all of the other nucleotides tested. The cross reactionwith PAPS is higher than expected given the lack of cross reaction withother adenine nucleotides. In this regard, it should be noted that mostcommercial PAPS preparations contain a significant fraction of PAP, butapplicants purchased HPLC-purified preparations that were analyzed atgreater than 95% purity, and took precautions in its storage and use toprevent hydrolysis. In any event, these results clearly show thatapplicants can generate antibodies that bind selectively to PAP in thepresence of PAPS, which is a key feasibility issue for allowingsufficient signal:noise and dynamic range in the proposed SULT assay. Anadditional 10-fold increase in Ab selectivity for PAP over PAPS may besufficient and is a very reasonable expectation using monoclonals ratherthan polyclonals.

[0207] The results observed with other antibodies and tracers testedwere similar. The additional antibodies produced from theN6-aminohexyl-PAP-KLH and 8-azido-PAP-BSA immunogens bound many of thetracers and the Ab/tracer complexes could be displaced in all cases byunlabeled PAP, indicating that the interaction was specific. The 1810antibodies showed very poor or no tracer binding and were not testedfurther. The polarization of the free tracers ranged from 15-40 mP andincreases observed upon Ab binding ranged from less than 100 mP toalmost 300 mP. Interaction with some tracers was too weak to allowsaturation with Ab, so maximal polarization changes were not alwaysobserved. More than ⅔ of the tracers tested bound to both types of Ab,but none of the tracers synthesized from N6-carboxymethyl-PAP bound toantibodies generated from either immunogen.

[0208] The FPIA-Based SULT Assay

[0209] Though applicants will need to produce an antibody thatdifferentiates between PAP and PAPS more effectively to develop a highquality assay, applicants were able to use the 3642 Ab and C8-PAP-F14tracer to monitor detection of PAP produced in reactions containing thepurified SULT1E1-cHis. In the initial experiment applicants sought toidentify the optimal PAPS concentration for maximal signal:noise.SULT1E1-cHis was incubated with estradiol and varying concentrations ofPAPS in the presence of pre-formed Ab-tracer complex; SULT1E1 waspresent at a level sufficient to rapidly drive the reactions tocompletion (FIG. 12).

[0210]FIG. 12 illustrates the effect of PAPS concentration on detectionof enzymatically generated PAP. The assay mixture included 200 ng ofSULT1E1-6×His (□) or assay buffer (▪) was added to wells containing 30mM phosphate (pH 7.4), 7 mM DTT, 8 mM MgCl₂, 75 mM NaCl, 0.5 mg/mL BGG,150 nM estradiol, 1 nM C8-PAP-F14 tracer, 12.5 uL Ab 3642, and varyingconcentrations of PAPS in a total assay volume of 100 μL. The plate wasincubated for 24 hour at 37° C. and read in a Tecan Ultra (Ex₄₈₅/Em₅₃₅).

[0211] As in a typical FPIA, the tracer was used at a concentration wellbelow the K_(d) and the Ab was adjusted to a concentration that resultedin approximately 85% of maximal tracer polarization in the absence ofcompetitor. FIG. 12 shows that there is a range of PAPSconcentrations—from approximately 1 to 5 mM—where the enzymaticallyproduced PAP causes a decrease in tracer polarization of approximately40 mP (the difference between the open and solid squares in FIG. 12).

[0212] That is, even though this antibody cross-reacts with PAPSsignificantly, it can be used in a competitive FPIA mode to detect PAPproduced in a SULT reaction with saturating PAPS (Km for PAPS withSULT1E1 is approximately 50 nM) with a dynamic range of 40 mP. Moreover,these were homogenous reactions, or single addition reactions, in whichall of the reaction and detection components were added at the start ofthe reaction, which is the preferred approach for an HTS assay. Similarresults are attained if the detection reagents are added after theenzymatic reaction is complete, thus the polarization signal is notaffected by the enzymatic reaction (data not shown).

[0213] Described above is an embodiment of how one of the Ab/tracerpairs applicants produced was used for detection of PAP in SULTreactions that are allowed to proceed to completion before reading.However, a continuous assay is the most desirable format for HTS becauseit allows accurate enzyme rate determinations and precludes the need fora quench step. FIG. 13 shows that the 3642 Ab and C8-PAP-F14 tracer canbe used to continuously monitor SULT1E1 enzyme activity over time,allowing determination of enzyme rates with diverse substrates.

[0214]FIG. 13 provides graphs of continuous FPIA-based detection of SULTactivity with diverse substrates acceptor substrates (200 nM) were addedto wells containing 30 mM phosphate (pH 7.4), 7 mM DTT, 0.8 mM MgCl₂, 75mM NaCl, 0.5 mg/mL BGG, 2 uM PAPS, 200 ng SULT1E1-cHis, 1 nM C8-PAP-F14Tracer, and 12.5 uL Ab 3642 in a total volume of 100 uL. Controlreactions (top trace in each graph) lacked C-His-SULT1E1 and containedall other reaction components. The plate was incubated at room temp andpolarization read at 1 minute intervals.

[0215] The top trace in each graph is a control reaction lackingSULT1E1, thus polarization does not change significantly, whereas in thereactions containing enzyme it decreases with time. The experimentalsetup was very similar to what might be done in an HTS setting: antibodyand tracer were put into the SULT reaction mix, dispensed into a 384well plate containing different acceptor substrates, and the reactionswere started by the addition of SULT1E1-cHis. The plates were then readat regular intervals and polarization values plotted as a function oftime. In reactions with known SULT substrates, the polarizationdecreased over time relative to control reactions lacking enzyme; as maybe expected as enzymatically produced PAP displaces the tracer from Ab.Note that there is no significant change in polarization when noacceptor substrate is present (graph f) or if SULT1E1 is absent (toptrace in each graph), indicating that the PAPS molecule is sufficientlyresistant to non-productive chemical or enzymatic hydrolysis.

[0216] These results show that a generic, fluorescence-based activityassay for sulfotransferases is technically feasible. In theseexperiments, the cross reaction of the 3642 Ab with PAPS contributessignificant background (i.e., decrease in polarization), limiting thedynamic range of the assay, but enzymatic rates can still be obtainedfrom the linear portions of the velocity curves. The bar graph in FIG.14 shows rates for each substrate calculated from linear portions of thevelocity curves in FIG. 13; though not a precise assay at this point,this rank-ordering of substrates does correlate inversely with theirpublished K_(m) values and with the substrate profile that applicantsdetermined using purified SULT1E1 and the ³⁵S-PAPS radioassay (FIG. 14).Accordingly, FIG. 14 is a comparison of SULT1E1 acceptor substrateprofiles determined using the FPIA-based assay and the ³⁵S-PAPSradioassay. Rates of FPIA-based reactions were calculated from linearportions of curves shown in FIG. 13. It is noted that the ³⁵S radioassaydata is provided in Table 4. Acceptor substrates were used at 200 nM(FPIA) or 400 nM (³⁵S-radioassay).

[0217] Lastly, applicants used a known SULT inhibitor, DCNP, to showthat the FPIA-based assay could be used to generate an inhibition curve(FIG. 15). Specifically, FIG. 15 is a graph showing inhibition ofSULT1E1 by 2,6 Dichloro-4-nitrophenol (DCNP) measured with theFPIA-based assay. DCNP was serially diluted two-fold into wells in 46 uLof phosphate assay buffer (30 mM KPO₄ (pH 6.5), 0.5 mg/mL BGG, 15 mMDTT, 1.6 mM MgCl₂, 4 μM PAPS), followed by 50 μl of a 2×Antibody/tracermix (5 uL 3642 Ab/2 nM Tracer C8-PAP-F14), and 200 ng of SULT1E in atotal volume of 100 μl. The plate was incubated at room temp for 30 min,and read on the Tecan Ultra. ΔmP values were calculated by subtractingthe SULT1E reactions from the no SULT1E controls. All values representthe mean of replicates.

[0218] The response of the FPIA assay to DCNP was validated bycomparison with the 35S-radioassay (data not shown); the K_(i) valuesdetermined with the two assay methods were 7.8 mM and 11 mM,respectively. Thus applicants have demonstrated that the assay can beused for detection of substrates and inhibitors—both of the key intendedHTS applications.

[0219] Taken together, these results clearly establish feasibility forproducing all of the components of the proposed FPIA based SULT assay:purified recombinant SULTs, antibody that selectively recognizes PAP inthe presence of PAPS, and fluorescent PAP tracers whose binding andcompetitive displacement from Ab can be detected by significant changesin polarization. In addition, applicants were able to demonstrate thatthe assay reagents could be used to detect SULT activity in a continuousmode, very similar to how the assay may be used in an HTS setting, thatthe response to different substrates is similar to the standardradioassay, and that the assay can be used for detection of aninhibitor.

[0220] Accordingly, it is envisioned that an antibody, suitably amonoclonal antibody with approximately 10-fold greater affinity andselectivity for PAP will be produced that will enable development of anassay with suitable dynamic range and signal:noise for commercial HTSapplications.

Example 4 Assay Systems

[0221] Although, the methods described herein may be utilized in avariety of different assay systems, in its simplest form, the presentassay system comprises an assay receptacle in which the assayed reactionis carried out, and a detector for detecting the results of thatreaction. In preferred aspects, the assay receptacle is selected from atest tube, a well in a multiwell plate, or other similar reactionvessel. In such cases, the various reagents are introduced into thereceptacle and typically assayed in the receptacle using an appropriatedetection system, described above such as a fluorescence polarizationdetector. In addition to a receptacle, a flat surface such as glass orplastic could also be used and the reaction components spotted onto thesurface in a defined array (such as a microarray).

[0222] Alternatively, and equally preferred is where the reactionreceptacle comprises a fluidic channel, and preferably, a microfluidicchannel. As used herein, the term microfluidic refers to a channel orother conduit that has at least one cross-sectional dimension in therange of from about 1 micron to about 500 micron. Examples ofmicrofluidic devices useful for practicing the methods described hereininclude, e.g., those described in e.g., U.S. Pat. Nos. 5,942,443,5,779,868, and International Patent Application No. WO 98/46438, thedisclosures of which are incorporated herein by reference.

[0223] In accordance with the above-described methods, it is envisionedthat an enzyme mediated coupling reaction between a first and secondreactant may be carried out in channels of a microfluidic device. Assuch, by using a microfluidics platform, it may be possible to mimic thecompartmentalization of a eukaryotic cell. This method could then beused to monitor the activity of group transfer reactions catalyzed byenzymes in a more native environment, in the context of other proteinsand with cellular components that may affect enzymatic activity.Therefore, data on the activity of enzymes that catalyze group transferreactions and the consequences of their inhibition can be obtained in asetting that will more accurately reflect an in vivo environment.

[0224] It is envisioned that these assay systems may be capable ofscreening test compounds that affect enzymatic reaction of interest.Optionally, devices used in accordance with the present invention areconfigured to operate in a high-throughput screening format, e.g., asdescribed in U.S. Pat. No. 5,942,443. In particular, instead ofdelivering potential test compounds to the reaction zone from areservoir integrated into the body of the device, such test compoundsare introduced into the reaction zone via an external sampling pipettoror capillary that is attached to the body of the device and fluidlycoupled to the reaction zone. Such pipettor systems are described in,e.g., U.S. Pat. No. 5,779,868 (fully incorporated by reference). Thesampling Pipettor is serially dipped into different sources of testcompounds which are separately and serially brought into the reactionzone to ascertain their affect, if any, on the reaction of interest.

[0225] Movement of materials through the channels of these microfluidicchannel networks is typically carried out using any of a variety ofknown techniques, including electrokinetic material movement (e.g., asdescribed in U.S. Pat. No., 5,858,195 (fully incorporated by reference),pressure based flow, axial flow, gravity flow, or hybrids of any ofthese.

Example 5 Assay Kit

[0226] Another embodiment of the invention is a kit for detecting andquantifying a Donor product of a group transfer reaction or a catalyticactivity generating the donor-product of a group transfer reaction. Thegeneral equation for the group transfer reaction includes adonor-X+acceptor→donor-product+acceptor-X, wherein the donor-product isdetected by the general detection reaction: firstcomplex+donor-product→second complex+displaced detectable tag. In itsmost simplest form, the kit for an FPIA immunoassay may include amacromolecule (i.e., antibody or an inactivated enzyme) and a tracer(displaced) and optionally the specific group transfer enzyme ofinterest. It is noted that the macromolecule and the tracer may eitherbe separate or incorporated into one solution vessel.

[0227] The kit may also include components such as, an activated donor,a detectable tag, acceptor substrates, inhibitors, buffers, cofactors,stabilizing agents, a set of instructions for using the kit, orpackaging and any combination thereof. In addition, the kit may beformatted for multiplex detection by using more than oneantibody/detectable tag pair where the detectable tags can bedifferentiated on the basis of the observables they produce. Theimmunoassay may be used to detect the donor product or the catalyticactivity generating the donor product.

[0228] In practicing the invention, it is encompassed that the kit maybe used for screening a library for a molecule or a set of molecules,capable of contacting an enzyme, wherein the enzyme generates thedonor-product in a group transfer reaction. The library may include atleast one of a plurality of chemical molecules, a plurality of nucleicacids, a plurality of peptides, or a plurality of proteins, and acombination thereof; wherein the screening is performed by ahigh-throughput screening technique using a multi-well plate or amicrofluidic system.

[0229] It is further envisioned that the macromolecule in the kitincludes at least one of an antibody, a polypeptide, a protein, anucleic acid molecule, an inactivated enzyme, and a combination thereofthat is capable of contacting the donor-product with high affinity. Itis further envisioned that the kit optionally include at least one of asulfotransferase, a kinase or an UDP-glucuronosyl transferase, a methyltransferase, an acetyl transferase, a glutathione transferase, or anADP-ribosyltransferase and combination thereof.

[0230] Although FPIA is a suitable mode of detection, also encompassedwithin the scope of the invention are kits designed to be used fordetecting donor product or the catalytic activity generating the donorproduct through other means such as a homogenous assay, a homogeneousfluorescence intensity immunoassay, a homogeneous fluorescence lifetimeimmunoassay, a homogeneous fluorescence resonance energy transfer (FRET)immunoassay or a homogenous chemiluminescent immunoassay, or anon-homogenous assay such as enzyme-linked immunoassay (ELISA). In thecase of a homogeneous fluorescence intensity immunoassay or ahomogeneous fluorescence lifetime immunoassay, the kit could be composedof an antibody and fluorescent detectable tag where the intensity and/orlifetime of the detectable tag is different when it is bound to antibodyand than it is free in solution. The difference in fluorescence; i.e.,the assay signal, could be enhanced by modification of the antibody suchthat its interaction with the detectable tag results in a further changein its fluorescence properties; i.e., quenching, enhancement, or achange in the lifetime. In the case of a homogenous FRET immunoassay,the interaction of a first fluor associated with the detectable tag witha second fluor that is attached to the antibody—either directly or viaassociated binding molecules such as biotin and streptavidin—couldresult in the excitation of the second fluor (or the reverse), therebygenerating a fluorescence emission at a wavelength different from thatof the detectable tag. The second fluor could be a small organicmolecule or a luminescent lanthanide probe. (It is noted that lanthanideemission is not fluorescence and is referred to as luminescence-basedresonance energy transfer, or LRET). In the case of chemiluminescentdetection, the detectable tag could be the donor product bound to onefragment of an enzyme used for chemiluminescent detection such asβ-galactosidase. When the donor product-fragment-one complex isdisplaced from antibody by the donor product, it would then bind tofragment two of the enzyme, producing an intact, active enzyme thatwould be capable of producing a chemiluminescent signal with anappropriate substrate. In the case of an ELISA, the assay would not behomogenous, and would require donor product or antibody be immobilizedto the surface of multiwell plates. A secondary antibody conjugated to adetection enzyme would also be included in this format.

[0231] All publications cited herein are hereby incorporated byreference in their entirety. In the case of conflict between the presentdisclosure and the incorporated publications, the present disclosureshould control.

[0232] While the present invention has now been described andexemplified with some specificity, those skilled in the art willappreciate the various modifications, including variations, additions,and omissions that may be made in what has been described. Accordingly,it is intended that these modifications also be encompassed by thepresent invention and that the scope of the present invention be limitedsolely by the broadest interpretation that lawfully can be accorded theappended claims.

We claim:
 1. A method of detecting a donor-product of a group transferreaction, the method comprising: a) reacting an activated form of adonor with an acceptor in the presence of a catalytically active enzyme;b) forming the donor-product and an acceptor-X; c) contacting thedonor-product with a first complex comprising a detectable tag capableof producing an observable; d) competitively displacing the detectabletag of the first complex by the donor product to generate a secondcomplex and a displaced detectable tag; and e) detecting a change in theobservable produced by the detectable tag in the first complex and thedisplaced detectable tag.
 2. The method of claim 1, further comprising,f) quantifying the observable of step (e).
 3. The method of claim 1,wherein, a) the activated form of the donor comprises donor-X; b) theacceptor comprises a substrate for the catalytically active enzyme,wherein the substrate is selected from the group consisting of apolypeptide, a protein, a nucleic acid, a lipid, a carbohydrate and asmall molecule substrate; c) the donor-product comprises a nucleotide ora non-nucleotide, wherein the non-nucleotide is a metabolic intermediateselected from the group consisting of s-adenosylhomocysteine,nicotinamide or coenzyme A; d) the acceptor-X comprises a reactionproduct in which X is a covalent adduct; wherein the covalent adduct isselected from the group consisting of a phosphate, a sulfate, acarbohydrate, a naturally occurring amino acid, a synthetically derivedamino acid, ADP-ribose, a nucleotide, a methyl, an acetyl, and aglutathione moiety; and wherein the covalent adduct is optionallycapable of altering either the function, the stability, or both thefunction and the stability of the acceptor; e) the first complexcomprises a macromolecule and a detectable tag; and f) the secondcomplex comprises the macromolecule wherein the detectable tag iscompetitively displaced by the donor-product resulting in the productionof an observable.
 4. The method of claim 3, wherein the macromolecule isselected from the group consisting of an antibody, a polypeptide, aprotein, a nucleic acid molecule, and an inactivated enzyme that iscapable of contacting the donor-product with high affinity.
 5. Themethod of claim 4, wherein the antibody is a monoclonal antibody, apolyclonal antibody, or a recombinant antibody.
 6. The method of claim4, wherein the antibody is specific for the donor product, and whereinthe level of antibody cross-reacting with the donor-X is less than thelevel of specificity that the antibody exhibits towards thedonor-product.
 7. The method of claim 1, wherein the detectable tag is atracer, wherein the tracer is a fluorescent or a chemiluminiscentmolecule conjugated to a nucleotide or a non-nucleotide.
 8. The methodof claim 1, further comprising detecting a catalytic activity, whereinthe catalytic activity generates the donor-product in the group transferreaction.
 9. The method of claim 8, wherein the catalytic activitycomprises a chemical catalytic activity, an enzymatic activity, or acombination thereof; wherein the enzymatic activity comprises asulfotransferase, a kinase, a UDP-glucuronosyltransferase, a methyltransferase, a acetyl transferase, a glutathione transferase, and aADP-ribosyltransferase.
 10. The method of claim 1, wherein the method isan immunoassay.
 11. The method of claim 10, wherein the immunoassay isselected from the group consisting of fluorescence polarizationimmunoassay (FPIA), fluorescence resonance energy transfer (FRET),enzyme linked immunosorbant assay (ELISA), chemiluminescenceimmunoassay.
 12. The method of claim 1, wherein the method is used forscreening a chemical library to identify a molecule which is capable ofactivating or inhibiting a group transfer reaction enzyme.
 13. Themethod of claim 12, wherein the molecule is capable of altering eitherthe function, the stability, or both the function and the stability ofthe acceptor.
 14. The method of claim 12, wherein the molecule iscapable of exhibiting a therapeutic effect.
 15. The method of claim 12,wherein the library is screened using a high-throughput screeningtechnique comprising a multiwell plate, a microarray or a microfluidicsystem.
 16. An antibody produced against a donor product of a grouptransfer reaction, wherein the antibody comprises the ability topreferentially distinguish between a donor-product and a donor in thepresence of a high donor concentration.
 17. The antibody of claim 16,wherein the donor-product is selected from the group consisting of anucleotide or a non-nucleotide.
 18. The antibody of claim 16, whereinthe antibody is specific for a phosphate portion of a nucleotide, andwherein the antibody has the ability to distinguish between a5′-phosphate, a 5′-phosphosulfate, a 5′-diphosphate and a5′-triphosphate.
 19. A homogeneous competitive binding assay for a donorproduct of a group transfer reaction, the assay comprising the steps of:a) combining the donor-product with a tracer and a macromolecule toprovide a mixture, the macromolecule being specific for the donorproduct, the tracer comprising the donor-product conjugated to afluorophore, the tracer being able to bind to the macromolecule toproduce a detectable change in fluorescence polarization; b) measuringthe fluorescence polarization of the mixture to obtain a measuredfluorescence polarization; and c) comparing the measured fluorescencepolarization with a characterized fluorescence polarization value, thecharacterized fluorescence polarization value corresponding to a knowndonor-product concentration.
 20. The assay of claim 19, wherein thegroup transfer reaction is catalyzed an enzyme.
 21. The assay of claim19, wherein the enzyme is selected from the group consisting of akinase, a sulfotransferase, a methyltransferase aUDP-glucuronosyltransferase, a acetyl transferase, a glutathionetransferase, and a ADP-ribosyltransferase.
 22. The assay of claim 19,wherein the donor-product is selected from the group consisting ofphosphoadenosine-phosphosulfate (PAP), adeno sine diphosphate (ADP),uridine diphosphate (UDP), s-adenosylhomocysteine, nicotinamide, andCoenzyme A.
 23. The assay of claim 19, wherein the fluorophore isselected from the group fluorescein, rhodamine, Texas red andderivatives thereof.
 24. A method of using the assay of claim 19 toscreen a chemical library to identify a molecule which is capable ofinhibiting or activating a group transfer reaction enzyme.
 25. An assaykit for characterizing a donor-product from a group transfer reaction,the assay kit comprising: a macromolecule and a tracer, each in anamount suitable for at least one homogeneous fluorescence polarizationassay for donor-product, wherein the macromolecule is a an antibody oran inactivated enzyme; and wherein the macromolecule and the tracer maybe separate or together in the container.
 26. The assay kit of claim 25,further comprising packaging, and instructions for using the antibodyand the tracer in the homogeneous fluorescence polarization assay, theantibody being specific for donor-product, the tracer comprisingdonor-product conjugated to a fluorophore, the tracer being able to bindto the antibody to produce a detectable change in fluorescencepolarization.
 27. The assay kit of claim 26 wherein the fluorophore isselected from the group consisting of fluorescein, rhodamine, Texas redand derivatives thereof.